Effects of Different Freezing Protocols on Motility, Viability, Mitochondrial Membrane Potential, Intracellular Calcium Level, and DNA Integrity of Cryopreserved Equine Epididymal Sperm.

The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processes 1 and 2) or a fast cooling rate (processes 3 and 4, respectively). Post-thaw total motility and slow sperm values were lower when using process 4 compared with processes 1 and 2 (P < .05) after 1 hour of incubation. Progressive motility was lower in process 4 compared with process 1 immediately after thawing and after 1 hour of incubation (P < .05). The amount of rapid sperm was lower when using process 4 compared with process 1 immediately after thawing (P < .05). After 1 hour of incubation, the amount of rapid sperm was lower when using process 4 compared with processes 1 and 2 (P < .05). Higher values for viable sperm were seen in processes 1 and 2 compared with process 4 (P < .05) after 1 hour of incubation. Immediately after thawing, more viable sperm with high MMP (hMMP) were observed when using process 3 compared with process 2 (P < .05). After 1 hour of incubation, a significantly higher amount of viable hMMP sperm were detected when using processes 1 and 2 compared with process 4 (P < .05). Process 2 yielded a lower percentage of sperm containing low calcium (lCa) than process 3 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of lCa sperm was observed using process 4 (P < .05). The subpopulation of viable/hMMP/lCa sperm was higher when using process 3 compared with process 2 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of this subpopulation was detected in process 4 (P < .05). The DNA integrity was similar in all groups. In conclusion, a slow cooling rate with a controlled rate freezer resulted in best sperm quality after thawing. Using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment is advantageous.

Comparison of the Effects of Five Semen Extenders on the Quality of Frozen-Thawed Equine Epididymal Sperm.

Cryopreservation of epididymal sperm allows the saving of genetic material in case of unexpected death or emergency castration. The aim of the present study was the comparison of five different combinations of extenders commercially available for equine frozen semen processing for cryopreservation of epididymal sperm. Epididymal sperm were harvested from gonads of 10 healthy stallions after routine castration by retrograde flush technique. Then, samples were split and diluted with (1) INRA96 + INRA Freeze, (2) BotuSemen + BotuCRIO, (3) EquiPlus + Gent Freeze, (4) EquiPlus + EquiPlus Freeze, and (5) Gent + Gent Freeze. Extenders 1 and 2 showed higher values for total and progressive motility after thawing compared with extender 4 (P < .05). Extender 3 was in between 1 and 2 (P > .05), and extender 5 resulted in the lowest values (P < .05). The subpopulation of viable frozen-thawed sperm with high mitochondrial membrane potential and low intracellular calcium content was higher using extender 1 compared with extenders 3, 4, and 5 (P < .05) and higher in extender 2 compared with extenders 4 and 5 immediately after thawing (P < .05). After 1 hour of incubation, this subpopulation yielded the highest values in extender 2 (P < .05). Immediately after thawing, extender 1 yielded higher values for percentage of DFI and mean DFI than extenders 3, 4, and 5 (P < .05). Following 1 hour of incubation after thawing, sperm processed with extender 1 resulted in the highest values for percentage of DFI and mean DFI (P < .05). Using extender 2, mean DFI values were lower than those in extender 1 and higher than the extenders 3, 4, and 5 (P < .05). The study revealed that according to the examined sperm quality parameters, freezing extenders (extender 1, extender 2) using low concentrations of glycerol either combined with or without methylformamide were beneficial for cryopreservation of stallion epididymal sperm. For processing of stallion epididymal sperm, an extender containing milk proteins (extenders 1-4) for initial dilution after sperm harvesting is preferable to an extender including egg yolk (extender 5).

[Birth of a foal following insemination with frozen-thawed epididymal sperm and simultaneous intrauterine delivery of homologous seminal plasma]. / Geburt eines Fohlens nach Besamung mit tiefgefrorenen Nebenhodenspermien und zeitgleicher intrauteriner Verabreichung von homologem Seminalplasma.

Cryopreservation of epididymal sperm allows final preservation of the gene reserve from valuable sires in case of unexpected injury terminating the breeding career. This case report describes the birth of a healthy foal following insemination with frozen-thawed epididymal sperm. The testes and epididymides were removed under general anaesthesia and sent cooled to the laboratory overnight. The cauda epididymidis was dissected and 17.79 × 109 sperm were harvested by a retrograde flush technique. A fertile mare was inseminated 1 year later with frozen-thawed epididymal sperm. Sperm were deposited into the tip of the uterine horn ipsilateral to the ovulation site. The mare did not become pregnant after the 1st cycle. In the 2nd breeding cycle, additional homologous seminal plasma was delivered into the uterus at the time of insemination and the mare was diagnosed as pregnant 14 days post ovulation. A healthy colt was born after 334 days of gestation. The method for preparing gonads for transportation to an appropriate laboratory is described for veterinarians and the different steps of semen collection and preservation are presented.

Fertility and 63,X Mosaicism in a Haflinger Sibship.

Chromosomal abnormalities are notable causes of infertility in horses. Mares show various degrees of estrous behavior, and ultrasound examination often reveals an underdeveloped genital tract. This article reports investigations on fertility in a Haflinger sibship with a healthy, normally developed, fertile mare with at least three healthy offspring. Chromosomal analysis performed incidentally and blinded for this mare revealed 63,X/64,XX/65,XXX mosaicism. Two closely related mares were also mosaics (63,X/64,XX), and one of them was a carrier of a marker chromosome. Repeated examinations of the mare and seven relatives (four mares and three stallions) did not provide evidence for sub- or in-fertility. They had no developmental abnormalities or conspicuous body conditions. Peripheral blood samples were collected for analysis of the karyotype and molecular analyses. Chromosomes were Giemsa stained and 4',6-diamidino-2-phenylindole banded to identify numerical or structural aberrations of chromosomes and identification of sex chromosomes, respectively. Fluorescence in situ hybridization was performed with an equine Y-chromosome painting probe to identify and count the sex chromosomes, and polymerase chain reaction analysis was used to test for the presence of the SRY gene and investigating chimerism. The present article demonstrates the necessity of further studies analyzing chromosomal X0 mosaics to improve the predictive value of chromosomal aberrations on fertility.

Postthaw Addition of Autologous Seminal Plasma Improves Sperm Motion Characteristics in Fair and Poor Freezer Stallions.

During semen processing for cryopreservation, most seminal plasma is usually removed, and components with protective effects on sperm may be missing after thawing and within the female reproductive tract. The present study evaluated the effect of postthaw addition of autologous seminal plasma on motion characteristics of stallion sperm with fair (n = 4) or poor (n = 3) freezability. Therefore, pure seminal plasma (group SP1), seminal plasma combined with fresh semen extender (group SP2), or seminal plasma mixed with freezing extender (group SP3) were used to fill 0.5 mL straws and frozen similar to stallion semen. Postthawing, semen samples (n = 42) were diluted either with semen extender (group FT) or with seminal plasma (n = 126) of groups SP1 to SP3 to 25 × 106 sperm/mL. In fair freezer stallions, total and progressive motilities were higher in group FT than in group SP1 (P < .05), but there was no difference in poor freezing stallions among groups (P > .05). However, comparing individual stallions, positive effects of seminal plasma on total or progressive motility were detected in two stallions. Curvilinear velocity increased in groups SP2 and SP3 in fair freezer stallions and in all groups with seminal plasma compared with group FT in poor freezer stallions (P < .05). Although straightness was higher in groups SP2 and SP3 compared with group FT in fair freezer stallions (P < .05), there was no difference among groups in stallions with poor freezability (P > .05). Average lateral head displacement did not change among groups of fair freezer stallions (P > .05) but was higher in groups SP2 and SP3 than in group FT in poor freezer stallions (P < .05). Beat cross frequency was higher in all groups diluted with seminal plasma postthawing in fair freezer stallions (P < .05), but only in group SP1 than in group FT in poor freezer stallions (P < .05). The addition of autologous seminal plasma to frozen-thawed semen can improve motion characteristics of stallions with fair and poor freezability. This is a valuable additional protocol for laboratories dealing with cryopreservation of stallion semen and for veterinarians working with fair or poor freezer stallions.

Disorder of Sexual Development in a Mare with an Unusual Tentative Mosaic Karyotype: 63,X/64,Xdel(Y).

The present report describes a 4-year-old Trakehner mare which was referred to the clinic for a breeding soundness evaluation. Clinical, histological, and postmortem examination revealed an underdeveloped genital tract, the absence of a cervix uteri, and small inactive ovaries without male gonadal tissue. Blood lymphocyte analysis revealed an unusual mosaic karyotype consisting of 2 cell lines. For the majority of cells (70%), monosomy X (63,X) was observed. The remaining cells (30%) contained 64 chromosomes including one X chromosome and a small rudimentary Y chromosome consisting mostly of heterochromatin. The centromere was retained, but its full functionality was questionable. PCR analysis revealed that the entire male-specific region of Y (Yq14), including the SRY gene, was deleted. It remained unclear if the pseudoautosomal region (Yq15) and parts of the heterochromatic region (Yq13) were affected by this deletion. The phenotype of the mare with this disorder of sex development associated with sex chromosome abnormalities is genetically comparable to 63,X monosomy which fully explains the clinical findings.

[Clinical signs in late pregnant mares]. / Klinische Parameter in der Spätträchtigkeit bei der Stute.

OBJECTIVE: During the peripartal period, interpretation of basic clinical signs may be challenging. In the present study, heart rate (HR), respiratory rate (RR) and body temperature (BT) were evaluated in healthy mares of different breed types and compared to reference values for adult horses from the literature. MATERIAL AND METHODS: During daily physical exams of periparturient mares, the HR, RR and BT were evaluated. Differences according to the horse's size were investigated and in large breeds, the influence of dystocia or retained placenta was analysed. RESULTS: During the last weeks before parturition (a. p.), the HR significantly increased and was clearly lower after parturition (p. p.; p < 0.05). In larger horses, the RR increased a. p. and decreased p. p. (p < 0.05). The BT underwent changes in all groups during the periparturient period and was higher p. p. (p < 0.05). In general, values for HR, RR and BT were highest in ponies (p < 0.05) while the lowest RR was measured in large horses (p < 0.05). There was no difference in the HR between mares with eutocia or with dystocia (p > 0.05). By contrast, the RR was significantly higher in mares with dystocia on day 1 p. p. (p < 0.05). Differences in the BT a. p. and p. p. occurred only in mares with eutocia (p < 0.05) and remained within the normal values. Mares with retained placenta did not exhibit significant changes in the HR (p > 0.05), but the BT was higher on day 1 p. p. (p < 0.05). CONCLUSION: Increased HR, RR and BT in mares during late pregnancy suggest a distinct physical performance for a prolonged period of time. Interpretation of these parameters in relation to the mare's reproductive state is essential to diagnose potential disorders and to determine whether therapy is required.

Alternative splicing after gene duplication drives CEACAM1-paralog diversification in the horse.

BACKGROUND: The CEA gene family is one of the most rapidly evolving gene families in the human genome. The founder gene of the family is thought to be an ancestor of the inhibitory immune checkpoint molecule CEACAM1. Comprehensive analyses of mammalian genomes showed that the CEA gene family is subject to tremendous gene family expansion and contraction events in different mammalian species. While in some species (e.g. rabbits) less than three CEACAM1 related genes exist, were in others (certain microbat species) up to 100 CEACAM1 paralogs identified. We have recently reported that the horse has also an extended CEA gene family. Since mechanisms of gene family expansion and diversification are not well understood we aimed to analyze the equine CEA gene family in detail. RESULTS: We found that the equine CEA gene family contains 17 functional CEACAM1-related genes. Nine of them were secreted molecules and eight CEACAMs contain transmembrane and cytoplasmic domain exons, the latter being in the focus of the present report. Only one (CEACAM41) gene has exons coding for activating signaling motifs all other CEACAM1 paralogs contain cytoplasmic exons similar to that of the inhibitory receptor CEACAM1. However, cloning of cDNAs showed that only one CEACAM1 paralog contain functional immunoreceptor tyrosine-based inhibitory motifs in its cytoplasmic tail. Three receptors have acquired a stop codon in the transmembrane domain and two have lost their inhibitory motifs due to alternative splicing events. In addition, alternative splicing eliminated the transmembrane exon sequence of the putative activating receptor, rendering it to a secreted molecule. Transfection of eukaryotic cells with FLAG-tagged alternatively spliced CEACAMs indicates that they can be expressed in vivo. Thus detection of CEACAM41 mRNA in activated PBMC suggests that CEACAM41 is secreted by lymphoid cells upon activation. CONCLUSIONS: The results of our study demonstrate that alternative splicing after gene duplication is a potent mechanism to accelerate functional diversification of the equine CEA gene family members. This potent mechanism has created novel CEACAM receptors with unique signaling capacities and secreted CEACAMs which potentially enables equine lymphoid cells to control distantly located immune cells.

Convergent evolution of pregnancy-specific glycoproteins in human and horse.

Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen cell adhesion molecule (CEACAM) family that are secreted by trophoblast cells. PSGs may modulate immune, angiogenic and platelet responses during pregnancy. Until now, PSGs are only found in species that have a highly invasive (hemochorial) placentation including humans, mice and rats. Surprisingly, analyzing the CEACAM gene family of the horse, which has a non-invasive epitheliochorial placenta, with the exception of the transient endometrial cups, we identified equine CEACAM family members that seem to be related to PSGs of rodents and primates. We identified seven genes that encode secreted PSG-like CEACAMs Phylogenetic analyses indicate that they evolved independently from an equine CEACAM1-like ancestor rather than from a common PSG-like ancestor with rodents and primates. Significantly, expression of PSG-like genes (CEACAM44, CEACAM48, CEACAM49 and CEACAM55) was found in non-invasive as well as invasive trophoblast cells such as purified chorionic girdle cells and endometrial cup cells. Chorionic girdle cells are highly invasive trophoblast cells that invade the endometrium of the mare where they form endometrial cups and are in close contact with maternal immune cells. Therefore, the microenvironment of invasive equine trophoblast cells has striking similarities to the microenvironment of trophoblast cells in hemochorial placentas, suggesting that equine PSG-like CEACAMs and rodent and primate PSGs have undergone convergent evolution. This is supported by our finding that equine PSG-like CEACAM49 exhibits similar activity to certain rodent and human PSGs in a functional assay of platelet-fibrinogen binding. Our results have implications for understanding the evolution of PSGs and their functions in maternal-fetal interactions.

Expression of enzymes and receptors of leukotriene pathway genes in equine endometrium during the estrous cycle and early pregnancy.

The aims of the present study were to elucidate the expression profiles of leukotriene (LT) pathway mRNA transcription and to determine the possible interaction of LT and prostaglandin (PTG) pathways genes in equine endometrium during the estrous cycle and early pregnancy. Endometrial biopsies were obtained from mares on the day of ovulation (d0), at late diestrous (LD, n = 4), and after luteolysis in the estrus phase (AL, n = 4) of the cycle. Biopsies were also taken on Days 14 (P14; n = 4), 18 (P18, n = 4), and 22 (P22, n = 4) during early pregnancy that were comparable days to cyclic sampling days. A mixed model was fitted on the normalized relative mRNA levels, quantified by qPCR in duplicate, and least significant difference test was employed to detect significantly different group(s). In addition, to determine the degree of contribution of each gene to separation of treatment groups, the multivariate projection method partial least square regression discriminant analysis was used. The expression of 5-lipoxygenase mRNA was greater on d0 and LD, declined at AL, and was suppressed by early pregnancy. Leukotriene A4 hydrolase mRNA expression increased at LD and during early pregnancy, but was significantly greater at LD compared with P14. The expression of LT C4 synthase mRNA was only induced at LD. Cysteinyl leukotriene receptors (CysLT1 and CysLT2) mRNA expressions were decreased by both cyclic changes and early pregnancy, whereas 5-lipoxygenase-activating protein and B leukotriene receptor mRNA expressions were not affected by early pregnancy or stages of the estrous cycle. Partial least square discriminant analysis suggests that LT and PTG pathway enzymes and receptors appear to behave similarly in terms of mRNA expression. In conclusion, the expression profiles of LT pathway genes are demonstrated in equine endometrium for the first time by the present study, and the present data suggest that LT pathway mRNA transcriptions are tightly regulated during early pregnancy in mares.

Exploration of global gene expression changes during the estrous cycle in equine endometrium.

The equine endometrium exhibits characteristic morphological and functional changes during the estrous cycle controlled by the interplay of progesterone and estradiol. A microarray analysis of endometrial tissue samples derived from five time points of the estrous cycle (Day [D] 0, D3, D8, D12, and D16) was performed to study the dynamics of equine endometrial gene expression. Statistical analysis revealed 4996 genes differentially expressed during the estrous cycle. Clustering of similar expression profiles was performed to find groups of coregulated genes. This revealed eight major profiles: highest mRNA concentrations on D0, from D0 to D3, on D3, from D3 to D8, on D8, from D8 to D12, from D12 to D16, and on D16. Bioinformatics analysis revealed distinct molecular functions and biological processes for the individual expression profiles characterizing the different phases of the estrous cycle (e.g., extracellular matrix and inflammatory response during the estrus phase, cell division and cell cycle during early luteal phase, and endoplasmic reticulum, protein transport, and lipid metabolism in the luteal phase). A comparison to dynamic gene expression changes in bovine endometrium identified common and species-specific gene regulations in cyclic endometrium. Analysis of expression changes during the estrous cycle for genes previously found to be differentially expressed on D12 of pregnancy provided new evidence for possible regulation of these genes. This study provides new insights regarding global changes of equine endometrial gene expression as molecular reflections of physiological changes in the cyclic equine endometrium with regard to the crucial role of this tissue for successful reproduction.

Influence of reproductive stage at PRID insertion on synchronization of estrus and ovulation in mares.

In the present study, we investigated the effects of reproductive status, size of follicles and plasma progesterone concentrations of mares at PRID insertion on the efficacy of the treatment, estrous cycle patterns, plasma concentrations of progesterone and LH. The progesterone-releasing device (PRID) was administered intravaginally to 28 Haflinger mares for 11 days at different reproductive stages: anestrus (n=6), estrus (n=11) and diestrus (n=11). Plasma concentrations of progesterone at insertion (Day 1) of PRID differed among treatment groups (anestrus: 0.2-0.6 ng mL(-1), estrus: 0.2-0.5 and diestrus: 1.6-10.8 ng mL(-1); P<0.001). Total secretion of progesterone (area under curve (AUC)) during treatment period revealed highest values in diestrus (38.2+/-3.1 ng mL(-1)h(-1)) followed by estrus (25.1+/-2.7) and anestrus (21.0+/-0.4 ng mL(-1)h(-1); P<0.05). Progesterone area under curve (AUC) was positively correlated with initial progesterone concentrations (R=0.5; P<0.05), but it did not correlate with the interval from PRID removal to ovulation. Plasma concentrations of LH during treatment period, were significantly lower in anestrous mares (184.6+/-28.6 ng mL(-1)h(-1)) when compared to estrous and diestrous mares (349.7+/-53.3 and 370.5+/-40.3 ng mL(-1)h(-1); P<0.05). Follicular size at PRID insertion had no effects on the intervals from PRID removal to subsequent estrus and ovulation. Follicle diameters at removal of PRID were significantly correlated with the interval from coil removal to estrus (R=-0.55, P<0.05) and ovulation (R=-0.72, P<0.0004) in cyclic mares. In anestrus 0 of 6 (0%) mares, in estrus 5 of 11 (45.5%) and in diestrus 6 of 11 (54.5%) mares ovulated within a defined interval of 1 day before to 1 day after mean interval from PRID removal to ovulation. In cyclic mares, response to treatment was significantly higher when compared to anestrous mares: almost all mares responded with estrus and ovulation independent from the stage of the estrous cycle at the start of treatment. However, accuracy of synchronization was still unsatisfactory. In cyclic mares, the plasma progesterone concentrations at insertion of PRID seem to be more important for the efficacy of the treatment than the assignment to estrous cycle stages.

Oxytocin does not contribute to the effects of cervical dilation on progesterone secretion and embryonic development in mares.

The aim of the present study was, to investigate the effects of oxytocin administration on Day 7 post-ovulation on progesterone secretion, pregnancy rate and embryonic growth in mares. Endogenous stimulation of oxytocin release was compared to the administration of native oxytocin or the long-acting oxytocin analogue carbetocin. At Day 7 after ovulation, mares had to undergo four treatments in a crossover design: (a) control, (b) oxytocin (10 IU i.v.), (c) carbetocin (280 microg i.m.) and (d) cervical dilation. On Day 13, all mares (8 of 8 mares) were pregnant on groups control, oxytocin and carbetocin and only 6 of 8 mares on group dilation. In one mare uterine fluid accumulation and uterine edema from Day 6 to 13 and early embryonic death by Day 11 occurred during dilation treatment. Another mare, which did not become pregnant during dilation treatment, developed uterine fluid accumulation and uterine edema from Day 10 to 14. Mean growth rates of the conceptuses did not differ among treatment groups and individual growth rates varied in a wide range from -0.1 to 0.8 cm per day. At Day 13, mean diameters of conceptuses yielded 1.4+/-0.1 cm in control group, 1.5+/-0.1 in oxytocin and carbetocin group and 1.3+/-0.2 cm in dilation group. Secretion of progesterone was not affected by treatments. Administration of oxytocin and carbetocin caused similar maximum plasma concentrations of oxytocin, but onset and duration of peaks differed. Maximum concentrations after intramuscular application of carbetocin were obtained almost 20 min later when compared to intravenous administration of oxytocin. Duration of peaks after injection of the long-acting oxytocin analogue was more than three-fold longer than after administration of native oxytocin. In conclusion, the present study showed that single administration of oxytocin or its long-acting analogue carbetocin at Day 7 after ovulation did not affect progesterone secretion, pregnancy rate and embryonic growth. Two possible scenarios concerning the effects of cervical dilation were observed: In the majority of mares, dilation of the caudal half to two-third of the cervical lumen up to a diameter of 4.5 cm had no negative consequences on progesterone secretion and pregnancy outcome. However, cervical dilation caused uterine inflammation and subsequent luteolysis in two mares and early embryonic death in one of them. Thus, manipulation of the cervix itself seems not to have negative impact on success rates of transcervical transfer of embryos in the mare.

Seasonal effects on attempts to synchronize estrus and ovulation by intravaginal application of progesterone-releasing device (PRID) in mares.

To investigate seasonal effects on the efficacy of estrus synchronization in mares, we administered a progesterone-releasing device (PRID) intravaginally to eight Haflinger mares for 11 days. In January 3 of 8 mares responded to the treatment with estrus and ovulation, in March 7 with estrus and 6 of 7 mares with ovulation, in June 6 of 7 and in October 7 of 8 mares with estrus and ovulation. Follicle distribution patterns at PRID insertion were different between January/October, March/June and June/October (P<0.05). Number of follicles decreased during PRID treatment in January, March and June (difference of number of follicles at Day 12 minus number of follicles at Day 1: -4.2+/-2.7, -0.9+/-0.9 and -4.9+/-1.5 follicles), while it increased in October (3.9+/-1.2 follicles; P<0.05). Mean progesterone concentrations were lowest in January (0.3+/-0.1 ng mL(-1)) when compared with March (3.5+/-1.8 ng mL(-1); P=0.063), June (4.4+/-1.4 ng mL(-1); P<0.05) and October (2.2+/-0.9 ng mL(-1); P<0.05). At Day 2 of PRID treatment, mean progesterone concentrations significantly increased in all mares. Except from January, mean LH concentrations decreased within one day after PRID insertion and remained at low levels during treatments in January and March. Total secretion of LH during PRID-treatment was significantly lower in January and March when compared with June and October. In the 5 of 7 mares that ovulated during PRID treatment a distinct increase of plasma LH concentrations after ovulation was detected. Administration of the progesterone releasing intravaginal device PRID combined with the PGF2alpha analogue cloprostenol was able to induce estrus and ovulation in mares at different times of the year. However, efficacy of the treatment was not satisfactory concerning effectiveness in relation to season and synchrony of intervals from removal of PRID to ovulation in mares.

Induction of abortion in queens by administration of cabergoline (Galastop) solely or in combination with the PGF2alpha analogue Alfaprostol (Gabbrostim).

The efficacy of cabergoline solely or combined with a PGF2alpha analogue in inducing abortion at different stages of pregnancy was investigated in 18 queens. The queens were assigned to two treatments: Group I ( n=10 )-cabergoline (15 microg/kg; daily, orally) and Group II ( n=8 )-cabergoline (15 microg/kg; daily, orally) combined with alfaprostol (10 microg/kg; every other day, subcutaneously). Each group was divided into two subgroups according to the duration of pregnancy when treatments started: Group IA ( n=8 ) included queens from Days 34 to 42 after mating. Group IB cats ( n=2 ) started treatments on Day 45 post-mating. Similarly, the combination of cabergoline and PGF2alpha analogue was first given to Group IIA ( n=6 ) from Days 25 to 40 of pregnancy and to Group IIB ( n=2 ) on Days 45 and 47, respectively. Termination of pregnancies was successful in all cats of Group IA, while treatments failed in both cats of Group IB, even though seven and eight treatments, respectively, were given. Mean (+/-S.D.) plasma progesterone concentrations before the start of treatments were 85.0+/-12.3 nmol/l and decreased within 3 days to 8 nmol/l and subsequently to basal values, when the queens aborted (Group IIA, n=6 ) or gave birth prematurely (Group IIB, n=2 ). When abortions failed (Group IB, n=2 ), progesterone concentrations remained elevated (16.9 and 9.8 nmol/l). Duration of combined therapy during late pregnancy in Group IIB ( n=2 ) lasted about 10 days. In both cases, premature birth occurred and the kittens died within 16 h after birth. Overall, treatments starting on Days 25-42 of pregnancy (Groups IA and IIA) had abortion rates of 100%. The average duration of treatments was 5.6+/-1.5 days (range, 3-8). Side effects seen were vomiting and occurred in 6 of the 109 (5.5%) treatments. In conclusion, pregnancies were successfully terminated in the second trimester of feline pregnancy by daily application of cabergoline solely or combined with the PGF2alpha analogue alfaprostol (given every other day). Cabergoline alone was ineffective in inducing abortion at later stages of pregnancy.

Estrous cycle characteristics, luteal function, secretion of oxytocin (OT) and plasma concentrations of 15-keto-13,14-dihydro-PGF2alpha (PGF2alpha-metabolite) after administration of low doses of prostaglandin F2alpha (PGF2alpha) in pony mares.

In the present study, the kinetics of the prostaglandin F2alpha (PGF2alpha)-metabolite 15-keto-13,14-dihydro-PGF2alpha after a single intramuscular application of various doses of the natural PGF2alpha dinoprost at Day 7 of the cycle in the mare were investigated. Effects of low doses on estrous cycle length and life span of corpus luteum were examined, because release of PGF2alpha is still under discussion to have detrimental influence on success rates of transcervical transfer of equine embryos. Eight Shetland pony mares were each randomly assigned to each of four treatments: (a) 0.8 mg/100 kg (group T1), (b) 0.4 mg/100 kg (group T2), (c) 0.2 mg/100 kg BM dinoprost i.m. (group T3), and (d) 1 ml physiological saline i.m. (group CO). Treatments were administered as single doses on Day 7 of the estrous cycle. Administration of dinoprost caused dose-dependent rises of plasma concentrations of PGF2alpha-metabolite, although values of individual mares showed great variation within groups. Prostaglandin treatments resulted in a distinct decrease of plasma progesterone concentrations to values between 1.6 and 7.9 ng/ml within 24 h. Treatment groups had significantly lower progesterone area under the curve (AUC: T1 942.8+/-175.9, T2 1050+/-181.2 and T3 1117+/-179.8 ng/ml/h) when compared with controls (CO 1601.9+/-227.6; t-test, P<0.05 ). There was a small, but significant negative correlation between AUC of progesterone and of PGF2alpha-metabolite ( R=-0.4; P=0.05 ). Administration of PGF2alpha caused secretion of oxytocin in three (T1, T2) and two (T3) mares out of eight ranging from 19.3 to 63.1 pg/ml. The AUC of oxytocin was positively correlated with AUC of PGF2alpha-metabolite ( R=0.4, P<0.05) and negatively correlated with AUC of progesterone ( R=-0.4, P<0.05). Administration of dinoprost yielded significantly shorter intervals from treatment to estrus and ovulation (values in parentheses), respectively, when compared with controls: T1 3.9+/-0.7 days ( 12.1+/-0.7 days), T2 4.5+/-0.6 ( 12.3+/-0.6 ), T3 4.9+/-0.5 ( 12.3+/-0.6 ), and CO 8.9+/-0.6 days ( 16.5+/-0.8 days) (t-test, P<0.01 ) (Fig. 2). Different doses of PGF2alpha caused similar effects. Data suggest that progesterone concentrations at applications influence efficacy of treatments more than doses administered, as demonstrated by their high correlation with estrous cycle patterns. It is important to note that differences we achieved are gradual and that all mares responded to treatment by luteolysis and premature estrus, regardless of doses applied.

Secretion patterns of oxytocin and PGF2alpha-metabolite in response to cervical dilatation in cyclic mares.

We conducted the present study to establish a standardized method for cervical stimulation without affecting the endometrium, and to investigate the effect on estrous cycle pattern and concentrations of progesterone, oxytocin and PGF2alpha-metabolite of cervical dilatation in the mare. Six healthy Haflinger mares underwent three different treatments (control, insertion, dilatation) on Days 5 and 7 of the cycles in different orders according to a Latin square design. During dilatation, the balloon of the catheter was inflated stepwise every 30s with warm physiological saline to a maximum of 50 ml. At this stage the size of the balloon was 4.5 cm in diameter and 6 cm length. Estrous cycle length was significantly shortened by dilatation when compared to controls (control: 22.8+/-1.7, insertion: 21.8+/-2.5, dilatation: 20.0+/-1.3 days; P<0.05). Concentrations of progesterone at Days 10, 12 and 14 after ovulation were significantly lower in dilatation cycles. Calculation of the area under the curve (AUC) for progesterone secretion from Day 7 to Day 12 also revealed a significant decrease in progesterone secretion in the dilatation group (dilatation: 34.1+/-7.3, insertion: 35.6+/-7.8, control: 39.1+/-5.9 ng/ml; P<0.05). Cervical insertion and dilatation caused a rapid and pronounced increase in plasma concentrations of oxytocin from basal levels (1.0-6.1 pg/ml) to maximum peaks (insertion: 125.5 pg/ml and dilatation: 305.2 pg/ml). The AUC for oxytocin was significantly higher after insertion (Day 5: 858.4+/-469.9; Day 7: 411.9+/-213 pg/ml/h) and dilatation (Day 5: 1697+/-1725; Day 7: 1078.5+/-764 pg/ml/h) when compared to controls (Day 5: 186+/-98; Day 7: 156+/-23.5 pg/ml/h; P<0.05). Manipulations did not cause considerable changes in plasma PGF2alpha-metabolite concentrations. Because cervical dilatation up to a diameter of 4.5 cm did not cause any immediate PGF2alpha release, the luteolytic pathway is unlikely to be responsible for shortening the length of diestrus and the estrous cycle. The present data suggest an involvement of oxytocin in the shortening of the luteal phase in response to cervical manipulation.