Induction of dog sperm capacitation by glycosaminoglycans and glycosaminoglycan amounts of oviductal and uterine fluids in bitches

Ejaculated sperm collected from 12 beagle dogs were incubated in canine capacitation medium (CCM), supplemented with 5 microg/ml chondroitin sulfate A (CS), 5 microg/ml hyaluronic acid (HA), or 5 microg/ml heparin (HP) for 7 hr at 38 degrees C in a 5% CO2 in air atmosphere to investigate the effects of glycosaminoglycans (GAGs) on dog sperm capacitation. The percentages of motile sperm, hyperactivated sperm (%HY), and acrosome-reacted sperm (%AR) in all media were examined after 4 hr and 7 hr of incubation. The oviducts and uteri of 9 anestrous and 18 estrous beagle bitches were removed under halothane inhalation anesthesia to measure the total GAG amounts in oviductal and uterine fluids. The lumens of the ampulla of the oviducts, isthmus of the oviducts, and the uterine horns were each flushed with 1 ml HEPES-EDTA fluid. Total GAG amounts in the flush fluids obtained were measured with a spectrophotometer. Sperm motility (51-59%), %HY (79-86%), and %AR (31-36%) in CCM supplemented with CS, HA, or HP were significantly higher after 7 hr of incubation than when incubated in CCM without GAGs (P<0.01 or 0.05). The mean total GAG amounts in the fluids from the ampulla and isthmus of the oviducts and the uterine horns in the estrous bitches were higher than in the anestrous bitches. These results indicate that GAGs in the oviductal and uterine fluids in estrous bitches are associated with in vivo sperm capacitation.     

Disappearance of the PHA-E lectin binding site on the surface of ejaculated sperm and sperm capacitation in the dog

Ejaculated semen and cross sections of the cauda epididymides collected from 3 normal dogs were smeared or stamped on glass slides, and the sperm on the slides were stained with 7 different FITC-lectins (Con A, DBA, GS-1, PHA-E, PSA, UEA-1, WGA) to examine the relation between sperm-binding glycoprotein derived from the canine prostate and sperm capacitation. The only lectin that stained the ejaculated sperm but not the cauda epididymal sperm was PHA-E. The sperm ejaculated by 5 other dogs were incubated for 4 hr in fluid flushed from the uterine horns or oviducts of estrous bitches, and then the percentages of actively motile sperm and hyperactivated sperm (HA-sperm) were determined. The percentages of PHA-E-labeled sperm and sperm labeled with fluoresceinated Ca indicator to assess the influx of Ca into the sperm were also evaluated. The mean percentages of actively motile sperm, HA-sperm, and Ca-labeled sperm after 4 hr of incubation in the uterine flush fluid and oviductal flush fluid were significantly higher than in control medium (P<0.05, 0.01), but the mean percentages of PHA-E-labeled sperm were lower (both P<0.01). The percentages of PHA-E-unlabeled sperm correlated with the percentages of both HA-sperm and Ca-labeled sperm (r(2)=0.787 and 0.812, respectively). The results indicate that loss of the glycoprotein secreted by the canine prostate on the sperm surface induces the influx of Ca into the sperm, and then hyperactivation of the sperm.     

In vivo capacitation and acrosome reaction of bovine sperm in estrous and diestrous cows

A staining procedure which enables distinction between spermatozoa possessing a true and false acrosome reaction (AR) was utilized to assess the incidence of capacitation and the true AR of bull spermatozoa recovered from the uterine horns of estrous and diestrous cows. Results show that at 3 and 6 h post-insemination, approximately 14.5 and 31.5%, respectively, of the live spermatozoa recovered had undergone a true AR in the uterus of estrous cows. An increasing percentage of those spermatozoa recovered from estrous cows with time were categorized as undergoing a false AR. This suggests that spermatozoa underwent capacitation, a true AR, then died prior to fixation and staining, therefore being grouped as false acrosome-reacted. Few spermatozoa were observed to have undergone a true AR in diestrous cows. It is apparent from this study that individual spermatozoa undergo capacitation and a true AR at different times during incubation in utero in estrous cows.     

Efficacy and toxicity of tamoxifen citrate for prevention and termination of pregnancy in bitches

Five groups of bitches were given tamoxifen citrate (1 mg/kg of body weight) orally twice daily for 10 days. Drug administration commenced during late proestrus, day 4 of estrus, day 2 of diestrus, day 15 of diestrus, or day 30 of diestrus (n = 4/group). Nineteen of the bitches accepted natural mating by 1 or more of 3 stud dogs of known fertility (1 bitch did not). Twenty days after cessation of drug administration, ovarian, uterine, and hepatic specimens were obtained from each bitch in 4 of the groups. Pregnancy proceeded to natural termination in bitches of the remaining group (diestrous day 30). Pregnancy was not detected in any bitch of the proestrus, estrous, or early diestrous groups. Of 4 bitches of each of the remaining groups (diestrous day 15 and diestrous day 30), 2 aborted fetuses and/or resorbed placental remnants; the other 2 bitches in each of these groups had normal-appearing fetuses (diestrous day-15 group) or clinically normal pups (diestrous day-30 group). Of the 20 bitches given tamoxifen citrate, 5 developed endometritis with or without pyometra, and 4 of these had ovarian cysts. Although tamoxifen citrate is effective for preventing or terminating pregnancy in the bitch, the regimen used in the study reported here was associated with high frequency of pathologic changes in the reproductive tract.     

Site of Intrauterine Artificial Insemination in the Bitch does not Affect Sperm Distribution within the Uterus

The aim of this study was to evaluate the distribution of frozen–thawed spermatozoa within the uterine lumen and oviducts following intrauterine laparoscopic deposition at two sites. Twelve bitches of unknown reproductive history were randomly distributed into two groups. Semen (3 ml containing 300 × 10⁶ frozen–thawed spermatozoa) was infused at the uterine body (UB group) or at the cranial tip of the left uterine horn. A 22‐G catheter was used to access the uterine lumen. Sperm cell distribution was evaluated after ovariohysterectomy performed 3 h after artificial insemination (AI). There was no difference between groups in mean time to perform AI. Spermatozoa were detected in all uterine segments, including the tip of both horns, but none was detected in the oviduct. The 22‐G catheter facilitated deposition of semen in the uterine lumen, particularly at the UB site. Sperm cell distribution occurred evenly along both horns, independent of the site of semen deposition.     

Influence of canine cumulus oophorus on homologous sperm motility

Sperm ejaculated by 8 beagle dogs and the cumuli oophori collected from 3 estrous beagle bitches were co-incubated, and penetration of the sperm into cumuli was observed to investigate the influence of cumuli on homologous sperm.The percentages of hyperactivated sperm and acrosome-reacted sperm were calculated after incubation with homogenized cumuli. The hyaluronic acid content of the incubated cumuli was measured, and hyperactivation and the acrosome reaction of the sperm were evaluated in medium containing hyaluronic acid. The mean percentage of hyperactivated sperm (33.0%) and number (3.0) of sperm that had penetrated a cumulus among sperm incubated for 7 hr were significantly higher than the values for sperm incubated for 0.5 hr (P<0.01). Almost all sperm that had penetrated the cumuli had intact acrosome, as though they were hyperactivated. The percentages of motile sperm (77.3%) and hyperactivated sperm (23.6%) after 2 hr incubation in the medium containing homogenized cumuli were significantly higher than in control medium (P<0.01), but there was no difference between cumulus and control media in the percentages of acrosome-reacted sperm. The hyaluronic acid content of a cumulus increased after 24 hr incubation. After 2 and 4 hr of incubation the percentages of hyperactivated sperm in the medium containing hyaluronic acid were significantly higher than in the control medium (P<0.01). These results suggest that canine hyperactivated sperm with intact acrosome can penetrate homologous cumuli and that the sperm are able to pass through the cumulus because the hyperactivated movement is maintained by hyaluronic acid secreted by the cumulus cells.     

Influence of Boar Seminal Plasma on the Distribution of Spermatozoa in the Genital Tract of Gilts

The main purpose of the present study was to investigate whether boar seminal plasma affects the transport of spermatozoa in the genital tract of oestrous pigs or not, with special reference to the sperm transport into the oviducts. Altogether 17 gilts were used in three experiments.Experiment I. In nine gilts one uterine horn was injected surgically with 10¹⁰ spermatozoa suspended in seminal plasma and the other uterine horn with 10¹⁰ spermatozoa suspended in TESNaK-glucose buffer solution. The sperm deposition was performed under general anaesthesia. The gilts were slaughtered 1–2 or 4–6 h after insemination. The genital tract was removed and the numbers of spermatozoa determined in oviducts and in uterine horns.Experiment II. The insemination doses were prepared exactly as in Experiment I. Approx. 24 h before insemination Polyvinylchloride cannulas were inserted into the uterine lumen of the horns, drawn via the midventral incision at linea alba subcutaneously to cutaneous incisions ventral to the vulva opening. One cannula was placed in each uterine horn. At standing heat the insemination doses were slowly injected through the cannulas. The gilts were slaughtered 1 h after insemination and the numbers of spermatozoa within the genital tract were counted.Experiment III. In three gilts under general anaesthesia the uterine horns were ligated 10 cm from the uterotubal junction. The semen doses (containing 2 × 10⁹ spermatozoa), prepared as in Experiment I, were deposited into the uterine horns anterior to the ligatures through a cannula. The gilts were slaughtered 1 h after insemination, and the numbers of spermatozoa within the oviducts and ligated part of the uterine horns were counted.In all three experiments more spermatozoa were, on average, recovered in the oviducts connected to uterine horns inseminated with spermatozoa suspended in seminal plasma. In Experiments I andII this was the case for 10 of 14 gilts and in Experiment III for all the three gilts. It is therefore suggested that boar seminal plasma pro¬motes sperm transport into the oviduct of oestrous pigs. The back¬ground mechanism for this is discussed.     

Induction of estrus and ovulation in the bitch, using exogenous gonadotropins

The capability of pregnant mare serum (PMS) and human chorionic gonadotropin (HCG) to induce estrus and ovulation was tested in mature, anestrous bitches. The PMS was given for 10 consecutive days in 1 of 3 regimens: 500 IU/day (experiment 1), 250 IU/day (experiment 2), or 20 IU/kg/day (experiment 3). The HCG was given as a single 500-IU dose on experimental day 10. Controls were given saline solution. Vaginal smears were collected on days 1, 3, 5, 7, 9, and 12 by jugular venipuncture, and the plasma was assayed for progesterone concentration by radioimmunoassay. On day 13, the bitches were euthanatized, ova were flushed from the uterine tubes (oviducts), and the ovaries were collected and prepared for microscopic examination. Fourteen of 25 bitches treated with PMS and HCG showed estrus and ovulated. Proestrus (vaginal bleeding) commenced between experimental days 7 and 10. Estrus commenced on day 9 or 10. Progesterone increased from approximately 1 ng/ml on day 1 to more than 6 ng/ml on day 12. Numbers of ovulation sites on both ovaries were 4.7 +/- 1.1 and 4.6 +/- 0.5 (mean +/- SEM) in those given the daily doses of 500 and 250 IU of PMS and 9.8 +/- 1.5 in experiment 3 bitches. Eleven hormone-treated dogs and 7 saline-treated dogs did not show any detectable response. Neither cystic nor unovulated, luteinized follicles appeared on the ovaries.     

Effects of hyaluronic acid on Ca(2+) influx, lactate dehydrogenase activity, and cyclic AMP synthesis in canine ejaculated sperm during In Vitro capacitation

The effects of hyaluronic acid, which comprises the cumulus intercellular matrix, on Ca(2+) influx, lactate dehydrogenase (LDH) activity, and cyclic AMP synthesis in canine sperm during capacitation was investigated. Ejaculated sperm were collected from 10 Beagle dogs and the sperm were incubated for 4 hr in Eagle's MEM containing 10 microg/ml of hyaluronic acid. The percentages of actively motile sperm, hyperactivated sperm (HA-sperm), acrosome-reacted sperm (AR-sperm), and sperm labeled with fluoresceinated Ca(2+) indicator (Ca(2+)-labeled sperm) were evaluated to assess Ca(2+) influx into the sperm. LDH activity and cAMP concentration were measured in homogenized sperm. The mean percentages of motile sperm, HA-sperm, and Ca(2+)-labeled sperm in the MEM containing hyaluronic acid were higher than in the control medium (P<0.05, 0.05, and 0.01, respectively), but there was no difference between the percentages of AR-sperm. Mean LDH activity and mean cAMP concentration were also significantly higher than the control values (P<0.05). The percentages of HA-sperm correlated with those of Ca(2+)-labeled sperm (r(2)=0.810). The results indicate that hyaluronic acid increases Ca(2+) influx, LDH activity, and cAMP synthesis in canine ejaculated sperm during capacitation in vitro.     

Sperm distribution in the reproductive tract of sows after intrauterine insemination

The purpose of the present study was to compare the number of spermatozoa obtained from different parts of the oviducts and the uterine horns of sows after intrauterine insemination (IUI) and conventional artificial insemination (AI), 24 h after insemination. Twelve crossbred (Landrace x Yorkshire) multiparous sows were used in the experiment. The sows were examined for standing oestrus using a back pressure test and were examined every 4 h after standing oestrus by real-time B-mode ultrasonography to estimate the time of ovulation. The sows were allocated to two groups, group I sows (n = 6) were inseminated by a conventional AI technique with 3 x 10(9) motile spermatozoa in 100 ml of extended semen, and group II sows (n = 6) were inseminated by an IUI technique using 1 x 10(9) motile spermatozoa in 50 ml of extended semen. A single dose of AI or IUI was given using the same boar, 8-10 h before the expected time of ovulation during the second oestrus after weaning. Twenty four hours after insemination, the sows were ovario-hysterectomized. The oviducts and the uterine horns were removed and divided into seven parts, the cranial, middle and caudal uterine horns, the utero-tubal junction (UTJ), the cranial and caudal isthmus, and the ampulla. All parts of the reproductive tract were flushed and the spermatozoa were counted using a haemocytometer. The results revealed that the spermatozoa were found in both the oviducts and the uterine horns in all animals. The number of flushed spermatozoa in the UTJ of groups I and II, was 142,500 and 131,167 (p > 0.05), and in the caudal isthmus was 1411 and 1280 (p > 0.05), respectively. The proportion of spermatozoa in different parts of the reproductive tract in relation to the total number of spermatozoa within the tract was not significantly different between groups I and II (p > 0.05). It could be concluded that IUI, with a three-time reduction in the number of spermatozoa used resulted in the same number of spermatozoa to be deposited in the sperm reservoir around ovulation time.     

Lectin-binding characteristics and capacitation of canine epididymal spermatozoa

Cross sections of the testes and the caput, corpus and cauda epididymides removed from 12 dogs were stamped on glass slides, and the sperm on the slides were stained with 6 different FITC-lectins (Con A, DBA, PNA, PSA, SBA, and WGA) to examine the characteristics of the surface glycoproteins (GPs) on canine epididymal sperm. The corpus epididymal sperm were washed three times by centrifugation, and their lectin-binding characteristics were investigated. The washed sperm from the corpus and cauda epididymides were incubated for 24 hr, and the fertilizing capacity of the sperm was evaluated by calculating the percentages of actively motile sperm (%MO), hyperactivated sperm (%HA), and acrosome-reacted sperm (%AR), and the number of canine zona-pellucida (ZP)-binding sperm. The testicular sperm did not stain with SBA lectin, but the SBA lectin fluorescence was observed on the surface of the entire heads of the caput epididymal sperm. Although all of the entire heads or acrosomal regions of the corpus epididymal sperm stained with all 6 FITC-lectins, the heads and acrosomal regions of the cauda epididymal sperm did not stain with DBA or SBA lectins. Washing the sperm from the corpus epididymis resulted in loss of the fluorescence of the FITC-DBA and -SBA lectins. The mean %MO, %HA, %AR, and ZP-binding number of the cauda epididymal sperm after 24 hr of incubation were higher than the values for the corpus epididymal sperm. All of the mean values for the washed sperm from the corpus and cauda epididymides were higher than the values for the unwashed sperm from the corpus and cauda, and with the exception of %AR, the values from the washed sperm from the corpus epididymis were significantly higher (P<0.05, 0.01). The results indicate that DBA- and SBA-lectin-binding GPs on the surface of canine epididymal sperm are associated with the fertilizing capacity and may be decapacitation factors.     

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine^®, 10 mg/kg) was injected subcutaneously 5 days before ovariohysterectomy in the treatment group (n = 6); bitches without treatment served as a control group (n = 6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p < .05) and ERα mRNA increased (p < .01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p < .05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p < .01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.     

Use of pulsatile intravenous administration of gonadotropin-releasing hormone to induce fertile estrus in bitches

The pulsatile IV administration of gonadotropin-releasing hormone (GnRH) was evaluated as a method to induce fertile estrus in 8 anestrous Beagle bitches. Bitches received 1.25 micrograms of GnRH every 90 minutes for 11 to 13 days. Gonadotropin-releasing hormone was delivered by use of an automatic pump. Reproductive history was known for all bitches, 4 of which, on the basis of 3 or 4 preceding cycles, had an interestrous interval of 219 +/- 14 days (mean +/- SEM). Estrus induction was attempted during early anestrus in 6 bitches (ie, 148 +/- 10 days since the preceding estrus) and late anestrus in 1 bitch (ie, 260 days since the preceding estrus); another bitch had not had an estrous cycle for nearly 2 years before GnRH administration. Signs of estrus were seen within 16 days after the start of GnRH administration in the bitches with regular estrous cycles (group 1, n = 7), and within 23 days in the bitch (group 2) with prolonged anestrus. All bitches were bred, and 7 of 8 (87.5%) became pregnant, with a mean litter size of 4.5 +/- 0.75. A normal hormonal response pattern was observed in group-1 bitches--a peak increase in plasma estrogen concentration of 22.3 +/- 2 pg/ml immediately before the onset of estrus. Peak plasma progesterone concentration (17.3 +/- 3 ng/ml) was observed 1 to 14 days after the onset of diestrus in the group-1 bitches that ovulated, and adequate plasma progesterone concentration was maintained throughout gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate

Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.     

Laparoscopic sterilization of the bitch and queen by uterine horn occlusion

Laparoscopic sterilization techniques, originally developed for use in women, were evaluated in the bitch and queen. In the first study (study I), the uterine horns of 6 bitches and 3 queens were occluded by electrocoagulation or plastic clips. The sites of occlusion were midway along the length of 1 cornus and at the uterotubal junction on the contralateral side. Both procedures effectively occluded the uterine horns, as evidenced by a distinctly visible separation of the reproductive tract. Laparoscopic examination 1 year after surgery revealed an enlarged, thin-walled, and fluid-filled uterine segment cranial to the midcornus occlusion sites in all animals. The contralateral horn was normal in appearance, except for the separation from the ovarian bursa. Three of the bitches developed pyometra (confined to the distended uterine segment) at 24 months, at 53 months, and at 72 months after sterilization, respectively. In a subsequent study (study II), 1 adult and 5 prepubertal bitches were sterilized by laparoscopic electrocoagulation of both uterine horns at the uterotubal junction adjacent to the ovarian bursa. Upon reexamination 1, 2, and 4 years later, the uterine horns of these females were normal in appearance, but were separated from the adjacent ovarian bursae. These females continued to be clinically healthy. Laparoscopic sterilization offers a rapid and safe alternative to ovariohysterectomy and, because of its minor invasive nature, can be performed on young, prepubertal animals. Such a procedure may have particular value as a simple, practical means of sterilizing dogs and cats on a mass basis.     

Distribution and viability of spermatozoa in the canine female genital tract during post-ovulatory oocyte maturation

Background

Unlike other domestic mammals, in which metaphase-II oocytes are ovulated, canine ovulation is characterized by the release of primary oocytes, which may take 12 to up to 36 hours. Further 60 hours are needed for maturation to secondary oocytes which then remain fertile for about 48 hours. Oestrus takes 7 to 10 days on average and may start as early as a week before ovulation. This together with the prolonged process of post-ovulatory oocyte maturation requires an according longevity of spermatozoa in the female genital tract in order to provide a population of fertile sperm when oocytes have matured to fertilizability. Therefore the distribution and viability of spermatozoa in the bitch genital tract was examined during post-ovulatory oocyte maturation.

Methods

Thirteen beagle bitches were inseminated on the day of sonographically verified ovulation with pooled semen of two beagle dogs containing one billion progressively motile spermatozoa. Ovariohysterectomy was performed two days later (group 1, n = 6) and four days later (group 2, n = 7). The oviduct and uterine horn of one side were flushed separately and the flushing’s were checked for the presence of gametes. The oviducts including the utero-tubal junction and the uterine horns, both the flushed and unflushed, were histologically examined for sperm distribution.

Results

The total number of spermatozoa recovered by flushing was low and evaluation of viability was limited. Prophase-I oocytes were collected from oviduct flushing in group 1, whereas unfertilized metaphase-II oocytes were detected in group 2. From day 2 to day 4 after ovulation a significant decrease in the percentage of glands containing sperm (P<0.05) and a marked reduction of the mean sperm number in uterine horn glands were observed. A concomitant diminution of spermatozoa was indicated in the utero-tubal junction accompanied by a slight increase in sperm numbers in the mid oviduct.

Conclusions

Oocyte maturation to metaphase-II stage is accompanied by a continuous sperm detachment and elimination in the uterine horns. Entrance of spermatozoa into the caudal oviduct seems to be steadily controlled by the utero-tubal junction thus providing a selected sperm population to be shifted towards the site of fertilization when oocyte maturation is completed.     

The influence of ovariectomy on luteinizing hormone concentrations in anestrous and cyclic sows

In an effort to determine whether anestrus in swine is due to aberrant ovarian feedback control of gonadotropin release, this study contrasted the influence of ovariectomy on LH concentrations in serum of anestrous sows and in sows that returned to estrus following weaning. Blood samples were collected at 6-h intervals from 7 d prior to until 4 d after ovariectomy of 22 anestrous and 24 cyclic sows. Blood samples also were collected at 15-min intervals for 8 h at 2 d prior to and 2 d after ovariectomy. Sampling at 6-h intervals continued until 12 d after ovariectomy and additional 8-h windows of 15-min samples were taken at 7 and 12 d after ovariectomy of seven anestrous and nine diestrous sows. Mean LH concentrations and LH pulse frequencies were greater (P less than .05) 2 d after ovariectomy than 2 d prior to ovariectomy in both anestrous and diestrous sows. Mean pulse amplitude had increased by 2 d after ovariectomy in anestrous sows but did not change in cyclic sows. Baselines as determined from the mean of all LH measurements excluding pulses, remained the same in both anestrous and diestrous sows at 2 d after ovariectomy. Pulse frequency, pulse amplitude, and mean LH concentration were greater (P less than .05) in both anestrous and diestrous sows at 7 and 12 d after ovariectomy than at 2 d prior to and 2 d after ovariectomy. Pulse amplitude on d 7 and 12 after ovariectomy decreased (P less than .05) in both anestrous and diestrous sows relative to those observed at earlier times.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vaginal and uterine microflora of adult dogs

Aerobic and anaerobic microflora were identified and quantitated in 82 vaginal and 78 uterine samples obtained from mature bitches during different stages of the estrous cycle. The mean +/- SD of total bacterial counts/100 mg of vaginal contents of the 82 bitches was log 5.0 +/- 1.5, ranging from log 2.4 to log 8.8. The count at the estrous stage (log 7.8 +/- 0.7) was significantly higher (P less than 0.05) than that at the anestrus (log 4.4 +/- 1.0), pregnancy (log 5.9 +/- 1.3), and postpartum (log 5.1 +/- 1.5) stages. The common organisms isolated from the vaginas were Bacteroidaceae, streptococci, Pasteurella spp, and mycoplasmas. Organisms were isolated from 48 (68%) of 78 uterine samples. The range of total counts/100 mg of uterine contents was from log 1.6 to log 8.3. Staphylococci and mycoplasmas were frequently isolated from the uterine contents. Although many uterine microfloras were similar to vaginal microfloras, some uterine culture had a single isolate identified. There were no pathologic findings in most of the uteri. Seemingly, vaginal bacteria frequently flow into the uterus, yet they rarely cause uterine infection.     

Influence of hormone supplementation to extended semen on artificial insemination,uterine contractions,establishment of a sperm reservoir,and fertility in swine

This study was performed to quantify the effect of hormone addition to semen using a low-fertility model to evaluate its effectiveness and mode of action. At 24 h after the onset of estrus, all gilts received a single low-dose AI (0.5 x 10(9) sperm/80 mL) with no hormone (control, C), estrogens (E, 11.5 microg), PGF2alpha (PG, 5 mg of Lutalyse), or oxytocin (OT, 4 IU), which were then evaluated for semen backflow (n = 48), oviductal and uterine sperm numbers (n = 28), uterine contractions (n = 12), pregnancy rate (PR, n = 120), and number of fetuses (n = 67). In Exp. 1, backflow of semen from the uterus was collected for 8 h after AI, whereas PR and fetuses were assessed at d 25 to 30 after AI. In Exp. 2, backflow was collected and reproductive tracts flushed to determine sperm numbers in the oviducts and the anterior segments of the uterus. In Exp. 3, sows were monitored for uterine contractions for 1 h before AI and for 2 h after AI. In Exp. 1, there was a treatment x time interaction for fluid loss (P < 0.001), but by 8 h after AI, there was no difference in the total volume (70 +/- 1 mL) of semen lost between hormone treatments (85%) compared to controls (90%). There was also a treatment x time interaction (P < 0.05) for number of sperm lost in the backflow (2.1 +/- 0.1 x 10(8)), but by 8 h following AI, there was no effect on total sperm lost for the hormone treatments (38%) compared to C (54%). There was a trend (P = 0.10) for increased numbers of sperm in the uteri of hormone-treated gilts (6.0 +/- 1.3 x 10(4)) compared with C gilts (2.2 +/- 1.3 x 10(4), but there was no effect of treatment on sperm numbers in the oviducts (3.2 +/- 1.3 x 10(4)). Within 0.5 h of AI, there was an increase in the frequency of contractions for PG compared with the other treatments (14.2 vs. 6.3/h, P < 0.005), however there was no effect on amplitude (54 mmHg) or duration (35 s) of contractions. The PR was not influenced by treatment and averaged 54% (P > 0.60), but total numbers of healthy fetuses were increased (P < 0.04) by PG (8.7) and tended (P = 0.06) to be increased for OT (8.4), but not for E (7.2) compared to C (5.8). Hormone addition to semen increased numbers of fetuses and this may be related to an alteration in the pattern of fluid and sperm loss after AI and a tendency for increased numbers of sperm in the anterior segment of the uterus. Therefore, in situations of lowered fertility, hormone addition could be a strategy to limit infertility in swine.