Comparison of the ovarian and uterine reproductive parameters, and the ovarian mRNA and protein expression of LHR and FSHR between the prepubertal and adult female cats.

This study aimed to evaluate and compare the ovarian and uterine characteristics along with the ovarian mRNA and protein expression of LHR and FSHR between the pre-pubertal and adult female cats. The uterine horns and ovaries were collected from pre-pubertal and adult female cats at their follicular, luteal and interoestrous stages of the oestrous cycle (n = 6/group). Endometrial and myometrial thickness, uterine gland diameter, ovarian weight and type of follicles were analysed. The mRNA and protein expression of LHR and FSHR was analysed by IHC and qPCR, respectively. The ovarian weight of pre-pubertal cats was significantly lower than that of adult cats. No differences were recorded in the numbers of primordial and primary follicles between the study groups, while adult luteal cats had significantly lower numbers of antral follicles compared to pre-pubertal cats. No differences in the ovarian expression of FSHR mRNA, LHR protein or mRNA were found between the pre-pubertal and adult cats, but significantly lower FSHR protein expression was found in pre-pubertal cats compared to adult luteal cats.

GnRH-agonist implants suppress reproductive function and affects ovarian LHR and FSHR expression in prepubertal female cats.

Effect of a GnRH-agonist (deslorelin) was studied on reproductive function and ovarian luteinizing hormone receptor (LHR) and follicle stimulating hormone receptor (FSHR) expression in prepubertal female cats that were either implanted with 4.7-mg deslorelin (implanted: n = 6) or not (controls: n = 18) or ovariohysterectomized at prepubertal age (prepubertal OVH: n = 6). Body weights, fecal estradiol, and sexual behavior of implanted and control cats were monitored for 48 weeks followed by collection of ovaries and uteri. Ovaries and uteri were collected from control cats at follicular, luteal, and inactive stage (n = 6/group) and from prepubertal OVH cats at prepubertal age. Ovaries and uteri were analyzed for anatomical/histological characteristics. Ovaries were also analyzed for LHR and FSHR expression. Statistical analysis showed higher (P ≤ 0.05) body weight in control than implanted cats only during 22nd to 26th weeks of the study. Estrus was observed in control cats only. Deslorelin reduced (P ≤ 0.05) ovarian weight and number of antral follicles but did not affect endometrial thickness and gland diameter. However, myometrial thickness of implanted cats was significantly lower than control cats at follicular and luteal stage. Ovarian LHR mRNA expression was lower (P ≤ 0.05) in implanted cats than control cats at follicular stage. FSHR mRNA and LHR protein expression did not differ among the three groups. FSHR protein expression was lower (P ≤ 0.05) in prepubertal OVH cats and was not affected by deslorelin. In conclusion, deslorelin suppresses reproductive function in prepubertal female cats for at least 48 weeks possibly through a change in the ovarian mRNA expression of LHR.

GnRH-agonist implantation of prepubertal male cats affects their reproductive performance and testicular LH receptor and FSH receptor expression.

This study was conducted to investigate the effect of GnRH-agonist implantation in prepubertal tomcats on sexual behavior, reproductive performance, and expression of testicular LH receptor (LHR) and FSH receptor (FSHR) and also to compare the testicular characteristics, LHR and FSHR expression between prepubertal and adult tomcats. In experiment 1, 3-month-old tomcats (n = 6/group) were either treated with or left without 4.7 mg deslorelin implants. Semen collection and evaluation were performed just before castration at 48 weeks after treatment; removed testes were analyzed for mRNA and protein expression of LHR and FSHR. We were able to collect semen from six non-treated cats, whereas in treated cats, semen was uncollectable. The results revealed that sexual behavior was absent in the implanted cats throughout the study period. Testicular volume was found to decrease from 30 weeks after treatment onward in the implanted cats compared to the controls (P < 0.05). Semen production was found only in non-implanted cats. Testicular tissue score, seminiferous tubule diameter, and LHR protein expression were found lower in the implanted cats (P < 0.05), but no differences were observed in mRNA expression of LHR and protein expression of FSHR between groups. The mRNA expression of FSHR was higher in the implanted (P < 0.05) compared to control cats. In experiment 2, testes from prepubertal (n = 6) and adult (n = 6) male cats were collected after castration and analyzed for mRNA and protein expression of LHR and FSHR. No differences were observed in the protein expression of LHR and FSHR between the two groups, whereas mRNA expression of FSHR was higher in prepubertal cats (P < 0.05). Testicular and epididymal weight, diameter of seminiferous tubules, and the testicular grade were higher in the adult compared to prepubertal cats (P < 0.05). In conclusion, deslorelin implants suppressed protein expression of LHR and enhanced mRNA expression of FSHR along with suppression of reproductive function without any adverse effects for at least 48 weeks in male cats.

The localization of Toll-like receptor 2 (TLR2) in the endometrium and the cervix of dogs at different stages of the oestrous cycle and with pyometra.

The aim of this study was to localize and evaluate the role of Toll-like receptor 2 (TLR2) in the endometrium and cervix of bitches at different stages of the oestrous cycle and in bitches with pyometra. Sixty-seven nulliparous dogs, ranging in age from 1 to 13 years, were allocated amongst five groups (pro-oestrus; n = 7, oestrus; n = 10, dioestrus; n = 16, anoestrus; n = 11, pyometra; n = 23). Blood samples were collected for the measurement of progesterone concentration. The mean progesterone concentration was analysed as a parameter for validating the stage of the oestrous cycle in bitches. Tissues collected from uterine horn and cervix were fixed in 4% paraformaldehyde for immunohistochemical examination of TLR2. The expression of TLR2 was assessed semi-quantitatively. No pathological changes were found in the uterine samples of healthy dogs. In bitches with pyometra, the glandular epithelium expressed TLR2 more intensely than the surface epithelium. The expression of TLR2 in the glandular epithelium was also significantly higher in healthy dogs at oestrus, dioestrus and dogs with pyometra compared with anoestrous dogs (p < 0.01). The expression of TLR2 in the stroma was not observed in the group of healthy dogs at all stages. The surface epithelium of cervix in dogs with pyometra expressed TLR2 significantly more intensely than did the stoma, whereas the expression of TLR2 during oestrus and dioestrus was absent in the stroma of cervix. This study provides the first report of immunohistochemical localization of TLR2 in the canine reproductive tract. In the present study, TLR2 was expressed in endometrial epithelium but was absent in the endometrial stroma of healthy dogs at all oestrous cycle stages. These findings suggest differential expression of TLR in endometrial cells. On the other hand, the lack of TLR2 in the stroma of healthy uteri of dogs may predispose to infection from the invading pathogens once the epithelial cells have been destroyed by the pathogens, especially Gram-positive bacteria.

Delay of puberty and reproductive performance in male dogs following the implantation of 4.7 and 9.4 mg GnRH-agonist deslorelin at an early pre-pubertal age.

CONTENTS: Stray dogs are a significant problem in large cities. Contraception is an important and useful solution to control the growing population of these dogs. Early-age neutering is an effective technique for canine population control; however, surgical neutering may not be possible in various situations. GnRH-agonist implantation has been successful for long-term reversible contraception in dogs. The efficacy of GnRH-agonist implantation on long-term suppression of reproductive performance was observed in male dogs. Eleven 4-month-old dogs were implanted with 4.7, 9.4 mg deslorelin or placebo. Sexual behaviour and testicular size were monitored every 2 months. Ejaculates were collected and evaluated at 8, 12, 15, 18, 24, 30, 32, 34 and 36 months of age. Dogs implanted with placebo were found to be healthy and in normal reproductive status. Most dogs (3/4) implanted with 4.7 mg deslorelin showed male sexual behaviour at age of 34 months old. From this group, two dogs had normal semen quality, while semen could not be collected from the other dog, and after castration, no sperm were obtained following epididymal flushing. One dog implanted 4.7 mg deslorelin and four dogs implanted with 9.4 mg deslorelin remained in the non-pubertal reproductive status at 30-34 months. The delay to puberty was longer in dogs implanted with higher dose of GnRH agonist. Implantation of pre-pubertal dogs with high doses of GnRH agonist will delay the onset of puberty and may be an effective strategy to reduce the number of unwanted breedings.

Differential expression of Toll-like receptor 4 (TLR4) in healthy and infected canine endometrium.

This study provides the first report into immunohistochemical localization of Toll-like receptor (TLR) in the canine reproductive tract. TLR4 was investigated in endometrium during the estrous cycle and in pyometra. Pyometra is the most important pathological condition of the uterus due to bacterial infection in dogs. To protect against invading pathogens, the female reproductive tract has evolved immune mechanisms. TLRs are the cellular components of the afferent arm of the innate immune system. The expression of TLR4 was significantly higher in the endometrial stroma compared to the endometrial surface epithelium and glandular epithelium in proestrus. The glandular epithelium and stroma at the diestrous stage expressed TLR4 significantly higher than surface epithelium. Furthermore, when compared to other healthy groups, the glandular epithelium at diestrus also higher expressed TLR4 than other stages. The expression of TLR4 in the surface epithelium was higher in dogs with pyometra compared with all other groups. And, the surface epithelium of dogs suffering from pyometra also expressed TLR4 more intensely than the glandular epithelium. The innate immunity of infected canine endometrium response to bacterial infection is intensely extremely increased by the expression of TLR4. Furthermore, the different levels of TLR4 expression seems related to physiological changes in distinct cell types of endometrium, leukocytes populations, cytokines and sex hormones.

Maturation competence of swamp buffalo oocytes obtained by ovum pick-up and from slaughterhouse ovaries.

This study was designed with the final goal of improving in vitro embryo production in the Thai swamp buffalo (Bubalus bubalis carabensis). Oocytes were collected by ovum pick-up (OPU) from six non-lactating multiparous swamp buffalo twice per week for 10 consecutive sessions followed by once-weekly collection for 10 consecutive sessions without hormone stimulation. In addition, oocytes were collected from slaughterhouse ovaries that were classified as follows: ovaries from non-pregnant cows with a visible corpus luteum (NPCL); pregnant cows with a corpus luteum (P); and non-pregnant cows without a corpus luteum (NP). Follicles in each group of ovaries were categorized as small (2-4 mm), medium-sized (5-8 mm) or large follicles (≥ 9 mm). The quality of the oocytes was assessed by their capacity to undergo in vitro maturation. The total number of observed follicles per session (all sizes combined) was larger in the once-weekly OPU group compared with the twice-weekly OPU group. In particular, the numbers of small and large follicles were higher in the once-weekly OPU group (5.2 ± 0.7 and 0.9 ± 0.2, respectively) than in the twice-weekly OPU group (3.9 ± 0.5 and 0.5 ± 0.1). The number of medium-sized follicles did not differ between the groups. The percentages of oocytes with an abnormal spindle morphology were not different between oocytes from the twice-weekly (30.0%) and the once-weekly (28.6%) OPU groups. A higher percentage of oocytes obtained in vitro (49.5%) exhibited nuclear abnormalities compared with those obtained in vivo (≤34.8%) after in vitro maturation. In conclusion, oocytes can be successfully collected by OPU in the swamp buffalo, without hormonal pretreatment, and per week more good-quality oocytes can be collected by twice-weekly OPU. In addition, oocytes collected from slaughterhouse ovaries can be used with the reproductive status of the cow having no influence on the maturation competence of oocytes.

Follicular dynamics and oestrous detection in Thai postpartum swamp buffaloes (Bubalus bubalis).

This study characterized follicular activity and oestrous behaviour from 5 to 9 days post-calving up to the 4th ovulation postpartum (pp) in 16 multiparous (range 2-7 parities) Thai swamp buffalo cows (Bubalus bubalis), aged 4-12 years and weighing from 432 to 676 kg. Ovarian follicular activity was examined by transrectal ultrasonography (TUS) every morning. Oestrous detection was performed twice daily by direct personal observation of behaviour and for presence of clear cervical mucus discharge and indirectly by video camera recording during 21 h/day. A follicular wave-like pattern was present before the 1st ovulation leading to short oestrous cycles. Growth rates and maximum diameters of the ovulatory follicles did not differ between the 1st and 4th ovulations. However, growth rate for non-ovulatory dominant follicles (DF) before the 1st ovulation was lower than for the ovulatory follicle (p<0.05). In addition, the diameter of all ovulatory follicles (14.3 ± 0.46 mm, n=39) was significantly larger (p < 0.01) than those of the preceding last but one non-ovulatory DF (10.8 ± 0.20 mm, n = 5), but similar to the last preceding non-ovulatory DF diameter (12.92 ± 0.96 mm, n = 14). Short oestrous cycles were most common between the 1st and 2nd ovulations (93.75%, 15/16 cows, 10.2 ± 0.38 days) decreasing in prevalence thereafter (50%, 3/6 buffaloes, 12.0 ± 1.53 days). Oestrous signs were relatively vague around the 1st ovulation pp to become more easily detectable thereafter. This study suggests that properly fed swamp buffaloes could be mated successfully within 2 months pp, at their 2nd spontaneous ovulation, provided oestrous detection is at least performed daily at 06:00-08:00 hour.

Y-chromosomal variation confirms independent domestications of swamp and river buffalo.

Y-chromosomal variation in the water buffalo was analysed by sequencing of DBY, ZFY and SRY gene segments. A clear separation of the paternal lineages of the river and swamp types parallels the differences between their maternal lineages and nuclear DNA. Sequence divergence was found to be comparable to the divergence of taurine cattle and zebu, and this divergence predated domestication, confirming that river and swamp buffalo originated from different wild populations. Within a sample of 23 Thai swamp buffaloes, we identified four haplotypes with different geographical distributions, two of which were shared by Thai wild buffaloes.

Seasonal and breed effects on reproductive parameters in bitches in the tropics: a retrospective study.

OBJECTIVES: To investigate the influence of season and breed on reproductive parameters in bitches raised under tropical climatic conditions. METHODS: Over a seven year period, from 1998 to 2004, 310 oestrous periods of 53 bitches were observed. The dogs were of various breeds; dobermann (number of bitches/number of oestrous cycles) (n=2/19), German shepherd dog (n=35/211), Labrador retriever (n=14/68) and Rottweiler (n=2/12). In 250 of the 310 oestrous periods, natural matings took place on days 9 and 11 after the onset of pro-oestrus. The whelping rate was analysed for bitches of each breed. Variables, including breed and the whelping rate, by month of the year, were used for analysis of the inter-oestrus interval, gestation length, total number of pups born, number of live pups born and the weight of the pups at birth. RESULTS: A low frequency of oestrous activity was found during the summer. Breeding dogs in the summer resulted in a low whelping rate. No difference (P>0.05) was seen in the whelping rate of each breed: dobermann (70.5 per cent), German shepherd dog (61.5 per cent), Labrador retriever (67.9 per cent) and Rottweiler (100 per cent). The Labrador retriever had a longer inter-oestrus interval (252 [114] and 190 [61] days) (P<0.01) and a larger litter size (8.2 [1.8] and 6.6 [2.8]) (P<0.05) than the German shepherd dog. CLINICAL SIGNIFICANCE: The environmental factors in summer tend to reduce oestrus incidence and fertility in the bitches. According to litter size, the Labrador retriever seems to have a more efficient reproductive performance than the German shepherd dog. The Labrador retriever had a longer inter-oestrus interval than the German shepherd dog.

Postpartum ovarian activity and serum estradiol-17beta level in Thai crossbred native mares.

To study the postpartum ovarian activities for investigation of first postpartum oestrus, twenty-five Thai crossbred native mares were monitored after parturition by oestrous detection, transrectal palpation and reproductive ultrasonography. Blood samplings were also taken for estradiol-17beta (E2) analysis. The first ovulation occurred within 20 days postpartum in 92% (23/25) of the mares. The mean intervals of foaling to first oestrus and to first ovulation were 10.3 +/- 2.9 and 13.4 +/- 2.6 days (mean +/- SD) respectively. Serum E2 increased from 7.0 +/- 2.9 to a peak of 10.8 +/- 3.3 pg/ml (mean +/- SD) at 2 days before ovulation. In conclusion, from the study, it can be stated that the postpartum breeding management should be considered after day 10 postpartum by careful examination of ovarian activity with various methods. However, the uterine condition should be also estimated associated with the ovarian activity after parturition which may increase breeding performance and foal production.

Induction of the acrosome reaction in dog sperm cells is dependent on epididymal maturation: the generation of a functional progesterone receptor is involved.

In the current study we investigated the progesterone receptor exposure on the sperm from the testis and different parts of the epididymis, the relation to the sperm maturation stage, the functionality of the progesterone receptor and the capacity of sperm to undergo acrosome reaction. Exposed progesterone receptors on spermatozoa were detected using Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) or a monoclonal antibody against progesterone receptor, C-262. Either progesterone or calcium ionophore was used to induce acrosome reaction. A high percentage (69 +/- 8%; mean +/- SD) of spermatozoa from the cauda epididymis showed P-BSA-FITC labeling at the onset of incubation, whereas only 0.1 +/- 1 and 4 +/- 2%, of spermatozoa from the testes, caput, and corpus epididymis, respectively, were labeled. There was no significant increase in P-BSA-FITC binding during the course of a 6 hr incubation. Treatment with either 10 microM progesterone or 5 microM calcium ionophore induced acrosome reaction in cauda epididymal sperm but not in testicular sperm, caput or corpus epipidymal sperm. It is concluded that the matured sperm of the dog from cauda epididymis and freshly ejaculated sperm demonstrate a functional membrane-bound progesterone receptor while less matured spermatozoa from the testicle, caput, and corpus epididymis fail to demonstrate such a receptor. Acrosome reaction of dog sperm can be induced using either progesterone or calcium ionophore; however, the maturation stages of spermatozoa influence this occurrence.

Effect of prostatic fluid on motility, viability and acrosome integrity of chilled and frozen-thawed dog spermatozoa.

Sperm preservation has become a routine procedure in dog breeding. In this study, the influence of prostatic fluid on sperm characteristics after preservation (either chilling or freezing) was investigated. The sperm-rich fractions of 20 ejaculates from five dogs were either extended without centrifugation or centrifuged and resuspended either directly in extender or in prostatic fluid before dilution with extender. Aliquots were processed for storage at 4 degrees C for 6 h or for freezing. Storage at 4 degrees C did not affect sperm motility, viability or acrosome integrity, irrespective of the dilution treatment. However, sperm motility and viability decreased significantly after freezing and thawing, particularly in the samples with additional prostatic fluid. In contrast, the acrosome morphology of viable spermatozoa was not affected by either the dilution method or by chilling or freezing and thawing. It is concluded that addition of prostatic fluid during semen processing adversely affects the motility and viability of frozen-thawed spermatozoa. However, prostatic fluid does not appear to affect the motility and viability of chilled spermatozoa or to alter acrosome integrity in either system of preservation.

Effect of sperm diluents on the acrosome reaction in canine sperm.

In this study we investigated the influence of sperm diluting media and temperature on the incidence of the acrosome reaction in dog sperm. Ejaculates were collected from 5 dogs, diluted with six different media and then incubated at 37 degrees C and 20 degrees C. Fluorescein isothiocynate conjugated peanut agglutinin (FITC-PNA) and ethidium homodimer as a vital stain were used in combination to determine the acrosomal status of viable spermatozoa, the technique was validated using electron microscopy. The outer acrosomal membrane of dog spermatozoa was shown to be the specific binding site for FITC-PNA. After 6 h of incubation, ejaculates diluted in media with a high Ca2+ concentration showed a significantly higher percentage (means +/- SD) of acrosome reacted spermatozoa [64 +/- 7 and 58 +/- 9 in sperm capacitation medium with (SP-TALP-1) and without BSA (SP-TALP-2), respectively] than those diluted in media with a low Ca2+ concentration [36 +/- 5, 39 +/- 4, 18 +/- 2 and 20 +/- 4 in Canine Capacitation Medium (CCM), Egg Yolk Tris dog semen extender (EXT-1), Modified Egg Yolk Tris extender (EXT-2) and Modified CCM (MCCM), respectively]. The increase in the percentage of acrosome reaction (AR) was slower at 20 degrees C than at 37 degrees C. In addition, the percentage of viable acrosome reacted spermatozoa increased significantly from 19 +/- 5 and 22 +/- 3 in non-bound sperm to 27 +/- 4 and 30 +/- 6 in zona pellucida bound sperm (diluted in EXT-2 and MCCM, respectively). We conclude that the composition of the spermatozoa diluent has a marked effect on the incidence of the acrosome reaction. Therefore, both the media used to dilute dog sperm and the temperature at which the spermatozoa are handled are important factors to consider when processing spermatozoa for artificial insemination, IVF procedures or preservation.

Detection of calcium ionophore induced membrane changes in dog sperm as a simple method to predict the cryopreservability of dog semen.

The sensitivity of dog sperm cells for extracellular Ca(2+)/Ca(2+)-ionophore challenge was compared to the detrimental effects of an optimized freeze/thawing protocol. Three sperm-rich fractions of ejaculates from 9 dogs were obtained, and one aliquot of each ejaculate was washed in a modified Tyrode's medium (HBT containing 0.1 mM Ca(2+)), without (control sample) and with 2.5 microM Ca(2+)-ionophore (induced sample) and incubated for 60 min at 38 degrees C in humidified atmosphere. Another aliquot from the same semen fractions was diluted, washed in a Tris buffer, and packed into 0.5-ml straws with a Tris buffer containing 7.5 vol % glycerol. The samples were stored for 1 week in liquid nitrogen after a computer-driven three-step freeze protocol and subsequently thawed for 50 sec in a 37 degrees C water bath and reconstituted into HBT. The acrosome integrity was determined using fluorescein-conjugated peanut agglutinin (PNA-FITC) as an acrosomal marker, while the vitality of the sperm cells was simultaneously assessed with the membrane impermeable DNA supravital stain ethidium homodimer 1 (EthD-1) using fluorescence microscopy and flow cytometry. The motility of frozen/thawed sperm samples was evaluated by microscopic as well as computerized motility analyses. Remarkably, the percentage sperm cells that underwent acrosome reactions induced by Ca(2+)-ionophore correlated very positively (r = 0.93) with the amount of acrosome damage observed in cryopreserved sperm samples. Furthermore, the degree of cellular damage induced by Ca(2+)-ionophore treatment correlated very negatively (r = -0.99) with the relative amount of sperm cells that remained motile after cryopreservation. Such clear correlations between Ca(2+)-ionophore induced acrosome reaction and motility parameters for frozen/thawed dog sperm cells were not found, suggesting that the generation of acrosome leakage and sperm immotility are two independent detrimental processes occurring during cryopreservation. From these results it can be concluded that Ca(2+)-ionophore treatment followed by simultaneous determination PNA-FITC and EthD-1 staining can be used to predict the cryopreservability of ejaculates from individual dogs used as donors.

Acrosome reaction in dog sperm is induced by a membrane-localized progesterone receptor.

The aim of this study was to investigate whether the dog sperm acrosome reaction can be induced by progesterone and whether the action of progesterone is mediated by binding of progesterone to a receptor on the sperm plasma. Progesterone-BSA conjugate labeled with fluorescein isothiocyanate (P-BSA-FITC) in combination with a vital stain, ethidium homodimer, was applied to visualize the presence of the progesterone receptor on living spermatozoa. Ten mM progesterone increased the acrosome reaction in viable spermatozoa over time from 3 +/- 1% at 0 hours to 69 +/- 8% at 6 hours (six dogs). In freshly ejaculated sperm from six dogs, P-BSA-FITC staining was observed in 13 +/- 1% of the viable, acrosome-intact cells, as characterized by bright fluorescence over the entire apical region. The proportion of P-BSA-FITC-stained, viable, acrosome-intact cells increased to 84 +/- 11% following 7 hours incubation in a low-calcium medium. In contrast, the majority (72 +/- 3%) of fresh epididymal sperm already demonstrated bright P-BSA-FITC staining. Apparently, epididymal spermatozoa already possess the progesterone receptor. The receptor is masked at ejaculation and subsequently gradually exposed.