Proliferative and apoptotic changes in the healthy canine endometrium and in cystic endometrial hyperplasia.

Proliferation and apoptosis play an important role in cyclic changes in the healthy canine endometrium. Proteins of the Bcl-2-family are known to be involved in the regulation of apoptosis. However, only few reports mention their expression patterns during cystic endometrial hyperplasia (CEH). In order to correlate proliferative and apoptotic processes, expression of the apoptosis-regulatory proteins Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) in healthy and cystic hyperplastic endometrial tissue as well as in pyometra was investigated. Uteri from 33 bitches were assigned to three groups: group 1 - healthy endometrium (n = 12), group 2 - CEH (n = 17) and group 3 - pyometra (n = 4). Markers for proliferation (Ki-67) and apoptosis (activated caspase-3 and TUNEL-method) as well as expression of Bcl-2 and Bax were determined in all endometrial layers. For groups 1 and 2 this was done during endometrial gland secretion in mid and late luteal phase (mLP, lLP), endometrial reparation in early anestrus (eAE) and in the regenerated endometrium in late anestrus (lAE). For group 3 only the late luteal phase was investigated. In group 1, cyclic proliferation patterns were found predominantly in superficial glands (SG) and stroma, whereas progesterone-mediated low expression levels coincided with high apoptosis rates in the basal glands (BG). In eAE, higher apoptotic activity was detected compared to lLP and lAE. Bcl-2 and Bax expression levels showed an inverse cyclic relationship in all tissue layers. In the stroma, in eAE, a rise in proliferative activity and concomitant increase in anti-apoptotic Bcl-2 expression levels was found, indicating that this layer serves as a source for endometrial regeneration. In CEH, no or limited cyclic patterns of proliferation, apoptosis, Bcl-2 and Bax were found. Increased proliferation rates coincided with deregulated apoptosis. Besides the glandular compartments, the stroma played an important role in the pathogenesis of canine CEH. In case of pyometra, both proliferation and apoptosis increased, indicating irreversible damage of the inflamed canine endometrium. In conclusion Bcl-2 and Bax play a role both in the physiological regenerative processes of the cyclic canine endometrium and in deregulating proliferation and apoptosis in CEH and pyometra.

Monochorial diamniotic dizygotic twins in a German Shepherd Dog: A case report.

CASE REPORT: A 6.5-year-old clinically healthy German Shepherd Dog with regular oestrous cycles of 6 months was presented for pregnancy diagnosis on day 38 after ovulation (p.ov.). Ultrasonography revealed three individual placental sites in progressed resorption and two vital adequately developed foetuses sharing a joint placenta. On days 41 and 48 p.ov., sonographic signs indicated normal development of both foetuses, but on day 52 p.ov., both foetuses were found to be dead. A caesarean section was performed the same day. Examination of the removed uterus confirmed the diagnosis of a "twin" pregnancy with two foetuses sharing the same placental site but separate amniotic membranes. One foetus showed generalized oedema (anasarca). Bacterial culture of swabs taken from inside the placental cavity was negative. At histological examination of the uterus, no signs of inflammation were found. Serum relaxin concentrations (day 38, 41, 48 and 52. p.ov.) were consistent with those of bitches with normal pregnancies. Cytogenetic analysis of the two foetuses revealed dizygotic twins, one male and one female according to SRY-PCR. By genotyping 17 high-polymorphic canine microsatellites, it could be demonstrated that the two foetuses developed from two different oocytes.

A commercial box for dog semen transport: What happens inside when the environmental temperature is increasing?

Environmental temperatures may influence the temperature inside commercial transport boxes during semen shipment and thereby storage conditions of diluted dog semen. To evaluate the temperature changes inside boxes and their influence on sperm quality, split semen samples (n=8) were placed in Neopor boxes(®) exposed for 48h to room temperature (RT) (Box 1), 40°C for 6h and then kept at RT (Box 2) or 40°C (Box 3). A fourth subsample was kept at 4-5°C in a refrigerator (control). Inside Box 1 temperature initially decreased to <3°C before it stabilized at 7-8°C, while in Box 2 no decrease occurred and temperature was at 7-8°C for 48 h. Temperature inside Box 3 was at 14-15°C for 24h and, thereafter, increased to 36.1°C. Analysis of sperm motility (CASA) and viability (PI and FITC-PNA) after 24 and 48 h revealed marked sensitivity of dog spermatozoa to temperature fluctuations (Box 1). A constant storage temperature of 7-8°C (Box 2) provided the most desirable semen quality in terms of motility, viability, as well as osmotic resistance when samples were stored for 48 h. Furthermore, results indicate that during 24h preservation a storage temperature of 14-16°C may provide optimum conditions for maintenance of sperm viability and function. An increase of the inside temperature to >30°C (Box 3) resulted in an almost complete loss in sperm integrity. In conclusion, results suggest a revision of current recommendations for storage temperature of diluted dog semen. Boxes for semen transport should be prepared depending on the expected environmental temperatures.

Effect of short-term scrotal hyperthermia on spermatological parameters, testicular blood flow and gonadal tissue in dogs.

The objective was to assess the effect of a short-term scrotal hyperthermia in dogs on quantitative and qualitative ejaculate parameters, testicular blood flow and testicular and epididymal histology. After a control period, the scrotum of seven normospermic adult beagle dogs was insulated with a self-made suspensory for 48 h. Nine weeks later, two animals were castrated, while in five animals, scrotal hyperthermia was repeated. Dogs were castrated either 10 or 40 days thereafter. In each phase of scrotal insulation, average scrotal surface temperature increased by 3.0°C. Semen was collected twice weekly throughout the experiment. Total sperm count did not change after the first hyperthermia, but it slightly decreased after the second (p < 0.05). Profiles of sperm morphology and velocity parameters (CASA) rather indicated subtle physiological variations in sperm quality than effects of a local heat stress. Chromatin stability of ejaculated spermatozoa as indicated by SCSA remained constant throughout the experiment. Perfusion characteristics of the gonads, that is, systolic peak velocity, pulsatility and resistance index at the marginal location of the testicular artery, did not change due to hyperthermia (p > 0.05). Histological examination of excised testes and epididymides for apoptotic (TUNEL and activated caspase-3) and proliferating cells (Ki-67 antigen) indicated only marginal effects of scrotal insulation on tissue morphology. In conclusion, a mild short-term scrotal hyperthermia in dogs does not cause substantial changes in sperm quantity and quality. In contrast to other species, canine testes and epididymides may have a higher competence to compensate such thermal stress.

[Clinical signs, diagnostic approach and therapy for the so-called ovarian remnant syndrome in the bitch]. / Klinische Symptomatik, diagnostische Vorgehensweise und Therapie beim so genannten Ovarrest-Syndrom der Hündin.

The ovarian remnant syndrome arises as a consequence of incomplete ovariectomy or ovariohysterectomy. Remnant ovarian tissue which has been left mostly unintentionally in the bitch may show endocrine activity a few weeks to several years after surgery, provoking a variety of clinical signs. The majority of affected bitches return to heat, in other cases signs of pseudopregnancy and endometritis may be observed. Occasionally, bitches with unclear clinical signs are presented with the suspicion of an inactive ovarian remnant. The following article intends to place the origin of the ovarian remnant syndrome into a factual context regarding the responsibility of the veterinarian and to demonstrate a reasonable diagnostic procedure according to the respective clinical signs. In this regard, the clinical-gynaecological examination, including vaginal cytology, must receive high priority, with the addition of progesterone analysis in peripheral blood plasma or serum if required. Using these combined diagnostic tools, ovarian remnants in stages of endocrine activity (follicular and luteal phases as well as cystic or tumourous ovarian tissue) can be easily unequivocally diagnosed. The application of a GnRH-stimulation test is only reasonable in bitches in which clinical signs are missing. In this context, the usefulness of semi-quantitative LH-assays is also discussed.

Apoptosis in the uterotubal junction and oviductal isthmus during the estrous cycle of the bitch.

Apoptotic cells were evaluated in the lamina epithelialis (LE) and lamina propria (LP) of the canine uterotubal junction and oviductal isthmus by means of the TUNEL assay and Caspase-3 detection regarding cyclic changes. Oviducts of bitches in anestrus, early and late follicular phase, 2 and 4 days after ovulation, mid-luteal phase, and endometrial reparation were available. The rate of apoptosis was generally low (min = 0; max = 33 apoptotic cells in 500 cells). Regardless of the stage of the estrous cycle, the number of apoptotic cells was significantly higher in the LE compared to the LP (P < 0.05). In anoestrus, early and late follicular phase as well as during endometrial reparation, the number of apoptotic cells was on a constantly low level. A significant increase was found 4 days after ovulation by caspase-3 detection coinciding with early luteal development and in mid-luteal phase. The results demonstrate that the number of apoptotic cells changes depending on the stage of the ovarian cycle. The coincidence of the increase in the number of apoptotic cells and the time of achievement of oocyte competence, which is known to occur in the bitch 3-4 days after ovulation, suggests a causal relationship between the postovulatory maturation stage of oocytes and the rate of apoptosis in the caudal oviduct. Furthermore, a specific relevance of apoptosis may exist in the process of fertilization regarding sperm storage and detachment of spermatozoa bound to the epithelium of the caudal oviduct, which has been identified as a functional sperm reservoir in other species.

Comparison of selected endocrine parameters during luteal phase and pregnancy in German Shepherd dogs and Beagles.

Concentrations of progesterone, prolactin and relaxin in serum at predetermined intervals after ovulation (day 0) in non-pregnant and pregnant normocyclic Beagles were assayed and results compared with those observed in German Shepherd dogs (GSD) in a previous study. The goal was to determine possible reproductive hormone specificities related to the GSD breed. Furthermore, the effects of medroxyprogesterone acetate (MPA)-treatment in non-pregnant Beagles and of progesterone supplementation in pregnant Beagles on the hormone concentrations were examined. Mean concentrations of progesterone and prolactin were not different in the non-pregnant Beagles compared with those seen in non-pregnant GSD, except at days 50-60, when progesterone concentrations were found to be higher in Beagles (p < 0.05). Mean progesterone concentrations in pregnant Beagles at days 50-60 after ovulation (day 0) were higher (p < 0.05) than in GSD at that time, but not at earlier time periods. Prolactin concentrations were higher (p < 0.05) in Beagles throughout pregnancy compared with those in the GSD. Mean relaxin concentrations were numerically but not significantly lower in GSD than in Beagles throughout pregnancy. A 10-day oral MPA treatment did not affect progesterone or prolactin secretion in normocyclic non-pregnant Beagles. Medroxyprogesterone acetate serum concentrations were approximately 3.9 ng/ml during treatment and decreased to 0.42 and 0.021 ng/ml within 5 and 15 days after end of treatment, respectively. Intramuscular progesterone supplementation from days 30 to 40 in pregnant Beagles resulted in higher concentrations of progesterone in the 36- to 45-day time periods; prolactin and relaxin concentrations were not significantly affected during or after treatment compared with administration of placebo. The results suggest a tendency towards deficient luteal function in the short-cycle GSD bitches previously studied, which in pregnancy may reflect the observed decreased prolactin concentrations; the possibility that GSD relaxin secretion is deficiency required needs further study. As oral treatment with MPA did not affect progesterone and prolactin release, it may be useful for studying luteal function in pregnant bitches with suspected hypoluteoidism.

Influence of progesterone withdrawal on pregnancy-related parameters during post-implantation early pregnancy loss.

Pharmacologically-induced luteolysis or treatment with an antiprogestin in early post-implantation pregnancy in dogs results in asynchronous death and resorption of conceptuses, indicating variable rates of response of individual conceptuses towards progesterone deficiency. This variability also seems to occur in bitches showing pregnancy failure in response to spontaneous luteal deficiency. In a total of 10 beagle pregnancies (two consecutive pregnancies of five bitches), abortifacient treatments beginning on day 24 after ovulation (ov) involved either administration of a progestin antagonist (total of six pregnancies, in three bitches) or a luteolytic regimen of prostaglandin F(2alpha)-analogue together with a dopamine agonist (total of four pregnancies, in two bitches). The outcomes were evaluated in relation to four control pregnancies in two bitches by assay of serum progesterone, prolactin and relaxin at selected time points or within selected time periods, by ultrasound of conceptuses including measurement of uterine blood flow, and parameters of the blood fibrinolytic system including plasma fibrinogen and plasminogen. The process of embryonic death and conceptus resorption was variable in onset and duration both in bitches that received the progesterone antagonist aglepristone (AGLE) and in those under the luteolytic treatment (cloprostenol combined with cabergoline). Pregnancy termination (death of all embryos or foetuses, respectively) occurred as early as day 29 and as late as day 41 after ov in AGLE-treated bitches, and not earlier than day 37 after ov in luteolytic-treatment bitches. Impending embryonic death was not predicted by changes in relaxin concentration, parameters of the fibrinolytic system, or in the perfusion of small uteroplacental vessels.

Morphological examination of the corpora lutea from pregnant bitches treated with different abortifacient regimes.

Different abortifacient regimes in dogs were analysed for their effect on the pregnancy corpora lutea (CL), namely, prostaglandin F2a analogue cloprostenol (CLO) combined with dopamine agonist cabergoline (CAB), or progesterone (P4) receptor antagonist aglepristone (AGL). Ovaries were collected after 6-10 days of treatment during first trimester. The CL of the control-group showed strong expression of relaxin (RLX), its receptor RXFP1 and enzymes of steroid biosynthesis (HSD) with high peripheral P4-levels. Whereas RXL, RXFP1 and HSD were lowest expressed in the CLO/CAB-group with a massive degeneration of CL and their blood vessels combined with low peripheral P4-level. The AGL-group showed less extensive CL degeneration and more intensive staining of the examined factors than CLO/CAB. In summary, all examined factors are associated with normal luteal function and are useful tools to stage luteolysis. Although both treatments have the same abortive action, their sequence of events on the CL is different.

Concentrations of prolactin, LH, testosterone, TSH and thyroxine in normospermic dogs of different breeds.

Concentrations of prolactin (PRL), LH, testosterone (T), TSH and thyroxine (T(4)) were determined before and at 20, 120 and 180 min after a single iv injection of thyrotropin-releasing hormone (TRH) in eight Beagles, eight Fox Terriers, six Labrador Retrievers and five Great Danes that were normospermic. Mean basal PRL concentrations were lower in the Fox Terriers compared with the Great Danes (p < 0.05). Mean LH concentrations were higher in the Fox Terriers than in the Beagles, and T was lower in the Fox Terriers at some times but not others (p < 0.05). Thyroid Stimulating Hormone (TSH) concentrations did not differ among breeds, while mean basal T(4) values were lower in Fox Terriers compared with Labrador Retrievers and Great Danes (p < 0.05). Stimulation of T(4) secretion 120 and 180 min after iv TRH injection was most pronounced in the Beagles and less in the Fox Terriers (p < 0.05). The results of the present study indicate that potential breed differences in circulating concentrations of PRL, LH, T, TSH and T(4) in male dogs with apparently normal fertility can be encountered, but further studies are needed to determine whether the observed differences are typical features of these breeds, reflect subsets of dogs within breeds, or are in part because of possible uncontrolled parameters such as sample timing, ambient photoperiod, housing conditions or diet.

Effects of short-term hyper- and hypoprolactinaemia on hormones of the pituitary, gonad and -thyroid axis and on semen quality in male Beagles.

Effects of a short-term hyper- and hypoprolactinaemia on serum concentrations of LH, testosterone and semen quality in six male Beagles were investigated. Blood samples were collected at 3-day intervals for 12 weeks. The time span was divided into five 3-week periods: pre-treatment, metoclopramide (MCP) treatment (0.2 mg/kg orally three times daily), cabergoline (CAB) treatment (5 microg/kg orally once daily), post-treatment 1 and post-treatment 2. In the latter, only semen characteristics were evaluated. Semen parameters were analyzed once per week during the whole 15-week investigation time. At the end of each period, the effects of a single intravenous injection of thyrotropin-releasing hormone (TRH; 10 microg/kg) on the secretion of prolactin (PRL), LH, testosterone, thyroid-stimulating hormone and thyroxine (T4) were investigated. Pre-treatment serum PRL concentration increased under MCP (p < 0.05), followed by a decrease under CAB administration (p < 0.05). Luteinizing hormone and testosterone concentrations were not affected. Except for straight-line sperm velocity, semen quality did not differ between collection periods. A single iv TRH injection induced a significant PRL increase at 20 min in all experimental periods except during CAB treatment. Luteinizing hormone and testosterone did not show clear TRH-related changes. Basic T4 levels were significantly reduced after CAB treatment (p < 0.05). The results of the present study demonstrate that MCP-induced short-term hyperprolactinaemia in male beagles does not seriously affect the hypothalamo-pituitary axis and semen quality.

Effects of gonadectomy on prolactin and LH secretion and the pituitary-thyroid axis in male dogs.

The effects of gonadectomy on the secretion of prolactin, LH, TSH, and thyroxine were investigated. Blood serum hormone concentrations were analysed before and at 20, 120, and 180min after a single iv TRH injection in each of eight healthy intact and castrated male beagle dogs before (control) and after 4-week treatment with the dopamine-2 receptor agonist cabergoline. Under control conditions the mean prolactin, TSH, and thyroxine concentrations were similar in intact and gonadectomised dogs, and administration of TRH provoked a significant (p<0.01) increase in concentrations of the three hormones. The overall inhibitory effect of cabergoline treatment on prolactin secretion was more pronounced in the castrated dogs compared with the intact group. Cabergoline significantly suppressed the TRH-induced prolactin increase in each group (p<0.01). Corresponding TRH-stimulated TSH concentrations were not affected by cabergoline. In the gonadectomised dogs, thyroxine concentrations before and at 120 and 180min after TRH injection were significantly lower than under control conditions. LH concentrations were always higher (p<0.01) in gonadectomised dogs compared with the intact dogs, but appeared to be affected neither by TRH nor by cabergoline administration. It can thus be concluded from the results, that gonadectomy does not result in hyperprolactinaemia in male dogs, while LH concentrations are significantly increased due to missing androgen feedback. Thyroid function remains unaffected by gonadectomy. Testicular steroids appear to interact with central dopaminergic and probably other neuroendocrine mechanisms regulating the secretion of prolactin, TSH, and thyroxine. Thus, long-term dopamine-2 receptor agonistic treatment may lead to a hypothyroid condition in castrated male dogs.

Prolonged foetal retention in a bitch resulting in trichogranulomatuous panmetritis and re-establishment of fertility after unilateral ovariohysterectomy.

This case report describes the clinical course, the diagnostic and therapeutic management as well as histopathological findings in a case of foetal retention for about 5 months in a 5-year-old Boxer. Clinical symptoms included mucosanguinous vaginal discharge and a firm ovoid mass of about duck egg size palpable in mid abdomen. Sonographic examination was not suitable to visualize uterine contents, while radiography revealed a conglomeration of foetal bones focally visible in the right uterine horn, which was surgically removed together with the ipsilateral ovary in order to maintain the fertility of the bitch. After lancing the uterine wall the osseous skeleton as well as parts of hairy skin and muscles of the foetus attaching the uterine wall were detected. Histologically a unilateral necrotizing panmetritis with almost complete destruction of the endometrial structures was diagnosed. Furthermore, the endometrium, the myometrium as well as the serosa revealed a severe trichogranulomatuous inflammation with numerous foetal hairs visible within the inflammatory alterations. After treatment of the bitch with prostaglandin F(2alpha) and an antibiotic agent she became pregnant in the second heat after surgery and gave birth to two healthy puppies. This is the first report on foetal retention for several months followed by unilateral ovariohysterectomy with subsequent recovery of fertility. Furthermore, this case report describes for the first time the histomorphological diagnosis of a unilateral trichogranulomatuous panmetritis in a bitch.

Determinants of sperm quality and fertility in domestic species.

Fertilization success cannot be attributed solely to the absolute number of vital, motile, morphologically normal spermatozoa inseminated into the female but more especially to their functional competence. A range of in vitro tests has therefore been developed to monitor crucial aspects of sperm function: their ability to adapt to changing osmotic conditions, to bind to the oviductal epithelium, and to undergo capacitation in an appropriate and timely manner. The tests employ flow cytometry in conjunction with fluorescent techniques, electronic cell counting, and computer-assisted image area analysis. The highly quantitative analysis provided by electronic sizing and flow cytometry enables assessment of representative cell numbers in a very short time with high reproducibility. More importantly, it allows the detection of physiological heterogeneity within an ejaculate in terms of the development of cell subpopulations and enables the kinetic analysis of changes in living cell suspensions. The tests offer a promising strategy for evaluating fertility in domestic animals. The capability for volume regulation ensures that sperm recover from the tonic shocks experienced at ejaculation and during cryopreservation. Assessment of capacitation in vitro provides valuable information on both the sperm's ability to respond to fertilizing conditions and the sequence and rates of ongoing capacitation/destabilization processes. The monitoring of response to capacitating conditions in kinetic terms allows the sensitive and adequate detection of sperm populations expressing fertilization attributes and their ability to respond to external stimuli in a timely manner. However, subfertility is likely to be associated with a suboptimal response (i.e. too high or too low) rather than a minimal response.

Morphology of canine placental sites after induced embryonic or fetal death.

Although spontaneous and medically induced canine embryonic or fetal death and "resorption" are clinically well documented, morphological studies of these processes are still missing. The objective of this study was therefore a detailed morphological investigation of canine placental sites after embryonic or fetal death. In five pregnant beagle bitches, embryonic or fetal death was induced by cloprostenol and cabergoline or by aglepristone. Two dogs served as untreated controls. Between Days 30 and 33 of gestation, the bitches were ovariohysterectomized, placental sites were fixed and examined by different methods. Morphological features of placental sites after both treatments were similar, finally leading to a complete disappearance of the placental labyrinth. Although there was an increase in the number of cells in the glandular chambers (superficial endometrial glands) expressing lysozyme after induced fetal death, signs of phagocytosis were absent in these cells, and no increased infiltration of maternal stroma by macrophages (compared to normal placental sites at the same time of gestation) occurred. We inferred that fetal and placental tissues were lysed, but no phagocytosis by genuine or "functional" macrophages was detectable. Further investigations are needed for a more detailed understanding of the morphological processes occurring after embryonic or fetal death in the dog.

Concentrations of progesterone, prolactin and relaxin in the luteal phase and pregnancy in normal and short-cycling German Shepherd dogs.

Twenty-two nonpregnant and 19 pregnant German Shepherd dogs were assigned to either a control group or a suspected short-cycling group, based on the interestrous interval (> or = 6 month and < 5 month, respectively) and data from previous pregnancies. Blood serum concentrations of progesterone and prolactin were determined from days 5 to 60 (day 0 = ovulation) for characterization of luteal function. In pregnant bitches, placental integrity was additionally assessed by relaxin concentrations. The nonpregnant, suspected short-cycling bitches had significantly lower progesterone concentrations than the controls, indicating decreased luteal activity both in the autonomous and prolactin-dependent period. In the pregnant suspected short-cycling bitches, unavoidable progesterone supplementation prevented assessment of luteal function; it may have suppressed prolactin secretion (significantly lower prolactin concentrations from days 20 to 60, compared with the pregnant control group), but deficient prolactin secretion affecting luteal function cannot be excluded. The significantly lower relaxin concentrations, together with a high incidence of embryonic death found in the pregnant, suspected short-cycling group, may indicate loss of placental integrity and may have caused decreased prolactin concentrations.

Influence of cooling rates and addition of Equex pasta on cooled and frozen-thawed semen of generic gray (Canis lupus) and Mexican gray wolves (C. l. baileyi).

A current priority for the preservation of the endangered Mexican gray wolf (Canis lupus baileyi) is the development of a sperm-based genome resource bank for subsequent use in artificial insemination. To optimize the quality of cryopreserved sperm, the procedures involved in processing semen before and during freezing need to be improved. The aim of this study were to examine the effects of: (i) different cooling periods before freezing and (ii) addition of Equex pasta (Minitüb, Tübingen, Germany) on the characteristics of sperm from the generic gray wolf and the Mexican gray wolf after cooling and cryopreservation. For Mexican wolf sperm, cooling for 0.5 and 1.0 h had a less detrimental effect on cell morphology than cooling for 2.5 h, whereas the slower cooling rate (2.5 h) had a less detrimental effect on functional parameters and seemed to cause less damage to plasma membrane and acrosome integrity than 0.5 and 1.0 h. For the generic gray wolf, cooling semen for 2.5 h had less detrimental effect on plasma membrane integrity and viability; together with the 0.5 h cooling time, it yielded the highest percentages of intact acrosomes. As previously shown in the domestic dog, Equex pasta had no beneficial effect on sperm characteristics in either wolf species.

Effects of the dopamine agonist cabergoline on the pulsatile and TRH-induced secretion of prolactin, LH, and testosterone in male beagle dogs.

In the present study, the pulsatile serum profiles of prolactin, LH and testosterone were investigated in eight clinically healthy fertile male beagles of one to six years of age. Serum hormone concentrations were determined in blood samples collected at 15 min intervals over a period of 6 h before (control) and six days before the end of a four weeks treatment with the dopamine agonist cabergoline (5 microg kg(-1) bodyweight/day). In addition, the effect of cabergoline administration was investigated on thyrotropin-releasing hormone (TRH)-induced changes in the serum concentrations of these hormones. In all eight dogs, the serum prolactin concentrations (mean 3.0 +/- 0.3 ng ml(-1)) were on a relatively constant level not showing any pulsatility, while the secretion patterns of LH and testosterone were characterised by several hormone pulses. Cabergoline administration caused a minor but significant reduction of the mean prolactin concentration (2.9 +/- 0.2 ng ml(-1), p < 0.05) and did not affect the secretion of LH (mean 4.6 +/- 1.3 ng ml(-1) versus 4.4 +/- 1.7 ng ml(-1)) or testosterone (2.5 +/- 0.9 ng ml(-1) versus 2.4 +/- 1.2 ng ml(-1)). Under control conditions, a significant prolactin release was induced by intravenous TRH administration (before TRH: 3.8 +/- 0.9 ng ml(-1), 20 min after TRH: 9.1 +/- 5.9 ng ml(-1)) demonstrating the role of TRH as potent prolactin releasing factor. This prolactin increase was almost completely suppressed under cabergoline medication (before TRH: 3.0 +/- 0.2 ng ml(-1), 20 min after TRH: 3.3 +/- 0.5 ng ml(-1)). The concentrations of LH and testosterone were not affected by TRH administration. The results of these studies suggest that dopamine agonists mainly affect suprabasal secretion of prolactin in the dog.

Volume regulatory function and sperm membrane dynamics as parameters for evaluating cryoprotective efficiency of a freezing extender.

In the past years a series of functional assays has been developed to determine the structural, morphological and functional integrity of the plasma membrane and sperm acrosomal membrane. Cell volume regulation is an important physiological function crucial for the success of cryopreservation. In this study, the effects induced by freezing-thawing were judged by evaluating the functional characteristics of frozen-thawed semen samples submitted to secondary stress such as osmotic challenge or incubation under capacitating conditions, following cryopreservation. Prior to freezing, dog semen samples were diluted in the presence or absence of Equex STM Paste, which contains sodium dodecyl sulphate (SDS) as the active ingredient. Cell volume regulation and capacitation and calcium ionophore-induced membrane dynamics were assessed in freshly diluted and frozen-thawed semen samples by electronic volume measurement and flow cytometry. Cryopreservation led to a disturbance of the volume regulatory function and to a rapid decrease in the proportion of acrosome-reacted live spermaotozoa. Extender containing Equex STM Paste had a protective effect on isotonic cell volume, on regulatory function under hypertonic conditions, and on the proportion of live acrosome-reacted cells. The evaluation of the functional state of sperm submitted to secondary stress after freezing-thawing leads to a more subtle characterization of sperm function and helps improve the cryoprotective efficiency of the extender.