Comparison of DNA Fragmentation Assay in Frozen-Thawed Cat Epididymal Sperm.

DNA fragmentation of frozen-thawed feline epididymal sperm from corpus and cauda regions was evaluated by three different techniques. The DNA fragmentation index (DFI) was compared between techniques: the sperm chromatin structural assay (SCSA(®) ), acridine orange staining techniques (AOT) and the sperm chromatin dispersion (SCD). There were significant differences in DFI among the techniques (p < 0.05) with no correlations. Only DFI values obtained from SCD revealed a significantly higher DFI in corpus compared with cauda spermatozoa (p < 0.05). The discrepancy between techniques might be due to the sensitivity of each technique, differences in severity of DNA damaged that can be detected. The difference in DFI between epididymal regions from SCD technique might indicate different maturational stages of spermatozoa, with less chromatin condensation of spermatozoa in corpus compared with cauda epididymis.

Cryopreservation of Dog Semen in a Tris Extender with 1% or 2% Soya Bean Lecithin as a Replacement of Egg Yolk.

Egg yolk is usually included in extenders used for preservation of dog semen. Lecithin is an interesting animal-protein free alternative to egg yolk for semen preservation. The aim of our study was to evaluate soya bean lecithin for cryopreservation of dog semen. Five ejaculate replicates were divided in three equal parts, centrifuged and each pellet diluted with one of the three Tris-based extenders containing 20% egg yolk, 1% soya bean lecithin or 2% soya bean lecithin. Extended semen was loaded in 0.5-ml straws, cooled and diluted a second time and frozen in liquid nitrogen vapours. Sperm motility parameters (CASA), acrosome integrity (FITC-PNA/PI) and sperm membrane integrity (C-FDA) were evaluated 5 min post-thaw and after 2 and 4 h of incubation. Total motility was significantly better in the egg yolk extender than in any of the lecithin-based extender and was better in the 1% lecithin extender than in the 2% lecithin extender. Sperm membrane integrity was significantly better in the egg yolk extender than in any of the lecithin-based extenders but did not differ significantly between the 1% and 2% lecithin extenders. Acrosome integrity was significantly better in the egg yolk extender than in the 2% lecithin extender but did not differ between the egg yolk extender and the 1% lecithin extender or between the two lecithin extenders. In conclusion, egg yolk was superior to lecithin in our study. The extender with 1% lecithin preserved sperm motility better than the extender with 2% lecithin.

Macroscopic and microscopic evaluation of Eurasian lynx (Lynx lynx) female tubular reproductive organs in relation to ovarian structures.

Although monitoring wild animals in the field is essential for estimations of population size and development, there are pitfalls associated with field monitoring. In addition, some detailed data about reproductive physiology can be difficult to obtain in wild live animals. Studying reproductive organs from the Eurasian lynx killed at hunting or found dead could be used as a valuable addition to other field data. We evaluated reproductive organs from 39 Eurasian lynx females (Lynx lynx) killed in Sweden during the hunting seasons in 2009, 2010, and 2011. According to notes on ovarian structures, the animals were categorized as being in one of four different reproductive stages: juvenile (n = 10), follicular stage (n = 8), luteal stage (n = 11), and anestrus (n = 10). Corpora lutea were classified as fresh CL from the present season or as luteal bodies from previous cycles. Microscopic evaluations were blindly coded while the outer measurements of the vagina and uterus were taken at the time of organ retrieval. The width of the endometrium, myometrium, outer width of the uterine horns, and the diameter of the vagina differed significantly with the reproductive stage (P < 0.001) and were largest in the follicular and luteal phases. The number of endometrial glands evaluated blindly coded on a subjective scale was significantly associated with the reproductive stage (P < 0.0001) and was significantly higher in the luteal phase than that in any other reproductive stages (P < 0.05). Cornification of the vaginal epithelium was only observed in females in the follicular stage or in females with signs of a recent ovulation. In conclusion, both macroscopic and histologic measurements are useful for a correct classification of the reproductive stage when evaluating reproductive organs in the Eurasian lynx killed during the hunting season. Routine evaluation of reproductive organs has a potential to be a useful additional tool to field studies of live lynx to monitor their reproduction.

Concentrations of anti-Müllerian hormone in the domestic cat. Relation with spay or neuter status and serum estradiol.

Female cats with unknown history can be diagnosed as spayed or intact with a GnRH-stimulation test or an LH test independent of the stage in the estrous cycle. However, although most females are correctly diagnosed with the LH test, the sensitivity and specificity are not 100%. The GnRH-stimulation test, although reliable, requires an injection of buserelin 2 hours before the blood sample is collected. Granulosa cells are the only cell type that produces anti-Müllerian hormone (AMH) in females, whereas Sertoli cells produce AMH in males. Anti-Müllerian hormone has been linked to spay status in dogs and cats and to ovarian and testicular pathology and fertility in different species. Our aim was to evaluate serum AMH concentrations in spayed female cats and in intact female cats of known age and reproductive stage (inactive ovaries or luteal phase). In addition, our aim was to compare serum AMH concentrations in intact and neutered male cats. We analyzed serum AMH concentrations in 15 spayed and 16 intact females and in 15 intact and 12 neutered male cats. Serum AMH was below the lowest standard point (<0.14 ng/mL) in all spayed females and neutered males, ranged between 1.3 and 19.0 ng/mL in the intact females and between 4.8 and 81.3 ng/mL in intact males. Thus, the AMH test had 100% sensitivity and specificity to diagnose the presence or absence of ovaries and testes in this study. In addition, in contrast to serum estradiol, serum AMH was not affected by buserelin stimulation (P = 0.459). Serum AMH was not correlated with serum estradiol before (rs = -0.188, P = 0.519) or after (rs = 0.335, P = 0.242) buserelin stimulation in the intact females. Four 6-month-old intact cats (two females and two males) had the highest AMH concentrations which in the females might represent a prepubertal peak previously described in other species and in males is likely due to high concentrations before puberty. In conclusion, we found that the AMH Gen II ELISA is reliable for diagnosing spay and neuter status of cats and that the domestic cat might be an interesting model for studies on AMH dynamics.

Effect of prostatic fluid on the quality of fresh and frozen-thawed canine epididymal spermatozoa.

Canine epididymal spermatozoa have a low freeze-tolerance ability compared with ejaculated spermatozoa, which could arise from the absence of prostatic fluid (PF). Therefore, the purpose of this work was to elucidate the influence of PF on the quality of canine epididymal sperm before and after freezing. Caudae epididymides were retrieved from eight dogs after routine castration. Spermatozoa were released by slicing the tissue and were extended in either Tris solution or PF before freezing. Frozen sperm samples were thawed at 70 °C for 8 seconds in a waterbath. Sperm concentration, motility using computer-assisted sperm analysis, morphology, plasma membrane, acrosome and chromatin integrity were assessed in the fresh sperm samples (after 20 minutes incubation) and at 0 and 4 hours after thawing. Progressive motility, distance straight line, distance average path, average path velocity, curvilinear velocity, straight line velocity, straightness, linearity, wobble, and beat cross frequency were significantly increased after extraction into PF. There was a higher proportion of spermatozoa with DNA damage in the PF treatment group at 4 hours after thawing than in the Tris treatment group (15.8% vs. 6.7%, P < 0.05). These results suggest that the addition of PF to canine spermatozoa activates sperm motility in fresh spermatozoa but has a negative effect on chromatin integrity after freezing-thawing.

Collection of field reproductive data from carcasses of the female Eurasian lynx (Lynx lynx).

Information about reproductive physiology in the Eurasian lynx (Lynx lynx) would generate knowledge that could be useful in the management of the Swedish lynx population based on the knowledge about their reproductive potential and population development. Age-related differences in ovulation and implantation rates would affect the reproductive output and the development of the population. The aims of this study were to evaluate a protocol for collection of reproductive data from carcasses by comparisons with published field data and to generate data about reproduction in the Swedish lynx. Reproductive organs from 120 females that were harvested between March 1 and April 9 from 2009 to 2011 were collected and evaluated macroscopically for placental scars. Females had their first estrus as yearlings but did not have their first litter until the next season. Pregnancy rates were lower in 2-year-old females than in females aged 3 to 7 years but did not differ significantly from females aged 8 to 13 years (54.5%, 95.6%, and 75.0%, respectively). CL from the present season were morphologically distinctly different from luteal bodies from previous cycles (LBPC). All females ≥3 years had macroscopically visible LBPC, whereas only 67% of 22 to 23 months old females had one to three LBPC and no females <1 year of age had LBPC. Females aged 34 to 35 months had up to eight LPBC, whereas the highest number of LBPC counted in females ≥3 years of age was 11. These data would be in agreement with only one estrus per season and LBPC from at least three previous reproductive seasons in older females. The number of LBPC was significantly correlated with the weight of the ovaries rs = 0.648, P < 0.001) and the age of the animals (rs = 0.572, P < 0.001). Uterine weight differed significantly with the stage of the reproductive cycle and was highest for mature females in the luteal phase of the cycle. The estrous period, defined as occurrence of ovarian follicles lasted from March 5 to April 1 in this material. In conclusion, this study confirms that useful information about lynx reproduction can be collected from reproductive organs retrieved after the death of the animals. Continuous monitoring of lynx reproductive organs would therefore make a valuable contribution to collection of field data, gathering information that can be useful for the management of lynx populations and potentially for the lynx as an indicator of environmental disturbances.

Comparison of the GnRH-stimulation test and a semiquantitative quick test for LH to diagnose presence of ovaries in the female domestic cat.

It is generally recommended that female cats not intended for planned breeding are spayed to reduce the population of feral cats and also because spaying is beneficial for the long-term health of the individual. For female cats of unknown origin or with estrous symptoms after spaying there is a need for a reliable method to diagnose or rule out the presence of ovaries to avoid unnecessary surgery. Methods previously recommended include vaginal cytology, evaluation of serum estradiol concentration during suspected estrus, induction of ovulation and subsequent evaluation of progesterone, or explorative laparotomy. These methods have the disadvantages that an accurate diagnosis only can be made during estrus or that an invasive procedure is required. Previously, the use of a GnRH challenge test and a semiquantitative LH test have been reported. Our aim was to compare these two methods. We therefore divided 31 female cats in two groups: (1) intact nonestrous females (N = 16), and (2) previously ovariohysterectomized females (N = 15). A blood sample was collected (Time 0) and 0.4 µg/kg buserelin (Receptal; Intervet, Danderyd, Sweden) was injected im. A new blood sample was collected 120 min after the injection. A drop of serum from the sample collected at Time 0 was placed on the LH test (Witness LH; Synbiotics, Corp., San Diego, CA, USA) and the result was evaluated as negative or positive. The remaining serum was frozen and analyzed for estradiol in one batch. Serum estradiol before buserelin stimulation ranged between 5 and 45 pmol/L (N = 14) in intact nonestrous queens and between 2 and 6 pmol/L (N = 15) in ovariohysterectomized females. Estradiol in samples collected after 120 min ranged between 12 and 51 pmol/L (N = 16) in intact queens and between 1 and 7 pmol/L (N = 15) in spayed cats giving a sensitivity and specificity of 100% for the buserelin stimulation test at a cutoff value of 11 pmol/L. All intact queens were negative in the semiquantitative LH test while 14/15 spayed cats were positive and one was negative giving a sensitivity of 100% and a specificity of 93.8% to detect the presence of ovaries in nonestrous cats.

Canine herpesvirus during pregnancy and non-pregnant luteal phase.

Canine herpesvirus (CHV) is a widespread infection among dogs that typically get latently infected after exposure and can reactivate the infection after stress. The aim of the present study was to study the effects of latent CHV infection during pregnancy on pregnancy outcome, and to study if there are signs of genital viral reactivation during pregnancy or during non-pregnant luteal phase. Twelve mated bitches and eight control bitches were followed and sampled regularly during pregnancy or non-pregnant luteal phase. Blood samples were taken for antibody analysis and vaginal swabs for real-time PCR analysis. Three of the pregnant bitches were vaccinated against CHV during pregnancy. All bitches had antibodies to CHV. Two pregnant bitches that were not vaccinated had a twofold or larger increase in CHV titre, with no negative effects detected on pregnancy. Higher titres were not associated with smaller litters or with vaccination. There was no consistent variation in antibody titres due to pregnancy or non-pregnant luteal phase. Vaginal excretion of CHV was not detected from any of the bitches.

The effects of antioxidants on semen traits and in vitro fertilizing ability of sperm from the flat-headed cat (Prionailurus planiceps).

Since antioxidants can overcome the negative effects of reactive oxygen species (ROS) during sperm cryopreservation, post-thaw sperm quality in flat-headed cats (Prionailurus planiceps), an endangered species, might benefit from the addition of antioxidants to semen extender. The objectives of this study were to: 1) investigate semen traits; and 2) evaluate effects of the vitamin E analogue Trolox (vitamin E) and glutathione peroxidase (GPx) on the quality of frozen sperm from captive flat-headed cats in Thailand. Eight ejaculates were collected by electroejaculation from four flat-headed cats. Each semen sample was divided into three aliquots and re-suspended in a semen extender as follows: 1) without antioxidant supplementation (control); 2) supplemented with 5 mM vitamin E; or 3) supplemented with 10 U/mL GPx. All samples were cryopreserved and thawed. Subjective sperm motility, progressive motility, and the integrity of the sperm membrane, acrosome and DNA were evaluated at semen collection, after 1 h cold storage, and at 0 and 6 h after thawing. Mitochondrial membrane potential, early apoptotic cells, and embryo development by heterologous in vitro fertilization were evaluated after thawing. Captive flat-headed cats were affected by teratozoospermia. After 1 h cold storage, sperm membrane integrity in samples supplemented with GPx was higher than the control group (54.5 ± 13.7 vs 51.3 ± 13.9; P < 0.05; mean ± SD). Sperm frozen in extender with GPx had higher motility at 6 h and greater mitochondrial membrane potential at 0 and 6 h post-thaw incubation than the other groups (P < 0.05). In conclusion, GPx improved the quality of frozen-thawed sperm in flat-headed cats.

Cryopreservation of epididymal cat spermatozoa: effects of in vitro antioxidative enzymes supplementation and lipid peroxidation induction.

Reactive oxygen species and lipid peroxidation reaction, causes of sperm damage, can be diminished by action of antioxidative enzymes. This study aimed to investigate effects of (1) the antioxidative enzymes; catalase, glutathione peroxidase and superoxide dismutase, on epipididymal cat sperm quality and (2) the lipid peroxidation reaction induced by a transition metal (ferrous ion (II); Fe(2+)) on sperm quality during the cryopreservation process. Epididymal spermatozoa harvested from 39 male cats were pooled and divided into 13 aliquots (n=13). Each aliquot was resuspended with either a Tris egg yolk extender I (control; EE-I), or the Tris egg yolk extender I supplemented with 200 U/mL catalase (EE-CAT), or 10 U/mL glutathione peroxidase (EE-GPx), or 600 U/mL superoxide dismutase (EE-SOD), and then cryopreserved. After thawing, each sperm sample was subdivided into two groups; with and without lipid peroxidation induction (EE-I plus Fe(2+), EE-CAT plus Fe(2+), EE-GPx plus Fe(2+) and EE-SOD plus Fe(2+)). Subjective sperm motility, membrane, and acrosome integrity were evaluated at the time of collection, after cooling, and at 0, 2, 4, and 6h after thawing. Motility patterns assessed by computer-assisted sperm analysis (CASA), mitochondrial activity, and DNA integrity were evaluated during post-thaw incubation, whereas percentage of lipid peroxidation was detected at 0 and 6h after thawing. The results demonstrate that catalase supplementation reduced linear motility and subjective motility immediately and 2h after thawing (P<0.05). Catalase supplementation, however, improved DNA integrity at 4h (P<0.05). Supplementation with glutathione peroxidase, compared to the control group, had a statistically significant positive effect on subjective motility at 0 and 6h, linear motility at 6h, mitochondrial activity at 6h, membrane integrity at 2 and 6h, and DNA integrity at 4h after thawing. Although superoxide dismutase had a positive effect on sperm membrane integrity at 2h after thawing (P<0.05), it significantly reduced membrane integrity after cooling, linear motility at thawing, and acrosome integrity at 2h after thawing. None of the three selected antioxidative enzymes significantly influenced acrosome integrity and none reduced the level of lipid peroxidation. Furthermore, induction of the lipid peroxidation reaction by Fe(2+) negatively affected most of the sperm quality parameters, i.e., motility and DNA integrity, during post-thaw sperm incubation (P<0.05). After thawing, there were, however, no significant differences between the control plus Fe(2+) and the antioxidative enzymes supplementation plus Fe(2+) groups. We can conclude that (1) glutathione peroxidase exhibits positive effects on post-thaw epididymal cat spermatozoa; but (2) none among the selected antioxidative enzymes could improve all sperm quality parameters; and (3) the lipid peroxidation reaction may be one cause of post-thaw epididymal sperm damage in cats, but the concentrations of antioxidative enzymes used in this study could not protect cat spermatozoa from lipid peroxidation induction.

Effects of thawing temperature and post-thaw dilution on the quality of cat spermatozoa.

The present study aimed to compare cat sperm quality after thawing using two different temperatures (37 and 70 degrees C) and to investigate the effects of post-thaw dilution on the sperm quality and longevity of ejaculated cat spermatozoa. Six ejaculates of each of six male cats were collected using an electroejaculator (total 36 ejaculates). The semen was frozen in 0.25-ml straws using a Tris egg yolk extender containing Equex STM paste. Four straws prepared from each ejaculate were thawed at four different occasions; (i) at 37 degrees C for 15 s, (ii) at 37 degrees C for 15 s and diluted 1 : 2 with Tris buffer (v/v), (iii) at 70 degrees C for 6 s, (iv) at 70 degrees C for 6 s and diluted 1 : 2 with Tris buffer (v/v). The percentages of motile spermatozoa, the scores of progressive motility, the percentages of spermatozoa with intact plasma membrane (using SYBR-14/EthD-1 stains) and intact acrosome (using fluorescein isothiocyanate conjugated peanut agglutinin/propidium iodide stains) were evaluated in fresh semen at 0, 2, 4 and 6 h after thawing. The thawing temperature had no effect on any sperm parameters throughout the incubation period (p > 0.05). The dilution after thawing improved sperm motility, progressive motility and acrosome integrity (p < 0.05). The thawing of cat spermatozoa and subsequently diluting with Tris buffer resulted in an immediate (at 0 h) overall (combined over temperature) percentage of motile sperm of 64.8 +/- 10.7 (mean +/- SD), a score of progressive motility of 4.0 +/- 0.5, a percentage of spermatozoa with intact plasma membrane of 64.4 +/- 12.1 and intact acrosome of 44.8 +/- 20.2. In conclusion, frozen cat semen can be thawed either at 37 or 70 degrees C and post-thaw dilution is recommended to reduce the toxic effect of some ingredients in the extender during post-thaw incubation.

Detection of lipid peroxidation reaction in frozen-thawed epididymal cat spermatozoa using BODIPY(581/591) C11.

Lipid peroxidation is considered to be an important cause of sperm membrane damage, resulting in an apparent reduction of reproductive fecundity. Recently, a new lipophilic fluorescent dye probe (BODIPY(581/591) C11; Invitrogen Singapore Pte Ltd, Singapore) has been demonstrated to be a highly sensitive indicator for the physiological oxidation of cell membrane fatty acids. The objectives of this study were: (i) to detect lipid peroxidation in frozen-thawed epididymal cat spermatozoa using the BODIPY(581/591) C11 and (ii) to study the effect of semen extender in protecting sperm membrane from lipid peroxidation [100-mm ferrous ion, ferrous sulphate (FeSO(4))]. Epididymal cat spermatozoa were collected from eight male cats. Two straws of sperm sample from each cat were cryopreserved. After thawing, the semen extender from the first straw was removed and the sperm pellet was resuspended with Tris buffer (control). The semen sample from the other straw was equally divided: one sample contained semen extender (treatment A) and one contained no extender (treatment B); both were incubated with FeSO(4). Semen samples were labelled with the BODIPY(581/591) C11 probe and evaluated by flow cytometry at 0 and 6 h after thawing (control), 6 h after the addition of FeSO(4) (treatment A), and 30 min and 6 h after the addition of FeSO(4) (treatment B), respectively. The percentage of lipid peroxidation was higher after treatment B (51.3 +/- 23.9) and 6-h incubation compared with the control and treatment A (p < 0.05). Furthermore, the percentage of lipid peroxidation after treatment B increased during the incubation time (p < 0.05). In conclusion, the high percentage of lipid peroxidation after treatment B indicated that FeSO(4) induced membrane damage in cat spermatozoa, which could be detected by BODIPY(581/591) C11. This marker is suggested to be a beneficial tool for the evaluation of lipid peroxidation. Furthermore, the use of semen extender seemed to protect cat spermatozoa membranes from lipid peroxidation.

Reproductive maturation in the male Eurasian lynx (Lynx lynx): a study on 55 reproductive organs collected from carcasses during 2002-2005.

Data on reproductive physiology from the Eurasian lynx (Lynx lynx) are still scarce. The lynx is protected under Swedish hunting legislation. All lynx that are found dead or that are culled at hunting are to be sent to the Swedish National Veterinary Institute. In this study, we examined reproductive organs from 55 male lynx collected during the years 2002-2005. Age, body weight, testicular weight and volume, production of spermatozoa, and sperm viability were evaluated. The majority of the animals (39) had been killed in February and March, which is during the hunting season. The ages varied between 6 months and 17 years, body weight between 3.6 and 25.5 kg, and mean testes weight between 0.16 and 3.16 g. The gonadosomatic index was low compared with other species (approximately 0.02% in mature males). Mean testes weight differed significantly between males <12 months of age and all other age groups but did not differ between males of 18-23 months and older males. Spermatozoa could be collected but had lost most of their viability. Seven of 10 males of 18-23 months were fertile, as defined by the production of spermatozoa while no males < or =15 months of age were fertile. Adherence of the prepuce to the penis and absence of penile spines were associated with immaturity. The results indicate that most males are fertile during the reproductive season of their second year.

Updates on reproductive physiology, genital diseases and artificial insemination in the domestic cat.

Reproduction in the domestic cat is characterized by large individual variations in the female oestrous cycle and in male semen quality. The female cat reproduction is strongly influenced by season, and new data suggest that it is possible that season also affects male fertility. Repeated periods of oestrus and spontaneous ovulation may lead to degenerative endometrial changes causing infertility. For artificial insemination (AI), induction of ovulation is necessary in the absence of the mating stimuli. The large variation in the relationship between follicular growth and timing of expression of oestrus complicates, however, timing of ovulation induction. Stress may lead to progesterone secretion by the adrenal glands and possibly have a negative impact on early pregnancy. The large individual variation in semen quality makes fertility evaluation and feline semen conservation a challenge. For AI, the semen can be deposited in the cranial vagina, the uterus or in the uterine tubes. Intrauterine insemination results in higher pregnancy rates than intravaginal but is more complicated. Surgical intrauterine or intratubal insemination has resulted in the birth of kittens, but is an invasive procedure that is not allowed in all countries. Transcervical intrauterine insemination with frozen-thawed semen has, however, recently resulted in the birth of kittens.

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa.

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.

Estradiol measurement after GnRH-stimulation as a method to diagnose the presence of ovaries in the female domestic cat.

There is currently no method to reliably diagnose the presence of ovarian tissue in inactive intact female cats except laparatomy which is an invasive procedure. All available tests require that the queen is in estrus. Obvious overt symptoms of estrus are, however, not always observed and some queens may have only 2 estrus periods/year. Therefore this study was designed to evaluate if it is possible to diagnose the presence of ovarian tissue by measurement of estradiol before and/or after stimulation with a GnRH-analogue. Twenty-two female cats were divided into two groups; 11 females that were known to have been ovariectomized and 11 females that were known to be intact. From each cat a heparinised blood sample was collected from the cephalic vein for resting estradiol and progesterone measurements. All cats were treated with a GnRH-analogue buserelin (Receptal, 0.4microg/kg im). Two hours later a second blood sample was collected. Median estradiol increased after stimulation with buserelin in intact but not in ovariectomized females (11 range 5-21 vs. 20 range 12-41, P=0.004 and 6 range 4-9 vs. 6 range 5-9 P=0.8) and did not overlap between the two groups. The highest estradiol concentration post-GnRH in the ovariectomized group was 9pmol/L while the lowest in the intact group was 12pmol/L. Progesterone was basal in all cats except one both before and after GnRH-stimulation. In conclusion this study demonstrates that measurement of estradiol concentration in plasma 2h after stimulation with a GnRH-analogue seems to be a reliable method to diagnose the presence of ovarian tissue in the female cat.

Pregnancy in the domestic cat after vaginal or transcervical insemination with fresh and frozen semen.

The objective was to compare pregnancy rates in domestic cats using fresh semen for intravaginal artificial insemination (IVI), either at the time of hCG treatment for induction of ovulation, or 28 h later, and to compare pregnancy rates following IVI or transcervical intrauterine insemination (IUI) of frozen-thawed semen. Eighteen queens were inseminated during 39 estrus cycles. Fresh semen with 13.5+/-5.4 x 10(6) sperm (range, 6.8-22 x 10(6)) collected by electroejaculation from four male cats was used in Experiment 1, and cryopreserved semen (20 x 10(6) sperm, with 70+/-5% post-thaw motility) from one male cat was used in Experiment 2. Serum concentrations of estradiol-17beta and progesterone were determined in most queens on the day of AI and again 30-40 days later. Treatment with 100 IU of hCG 3 days after the onset of estrus induced ovulation in 95% of treated queens. Pregnancy rates to IVI with fresh semen at the time of hCG administration versus 28 h later were not different (P=0.58); overall 33% (5/15) of the queens became pregnant. For frozen-thawed semen, AI was consistently done 28h after hCG administration; IUI and IVI resulted in pregnancy rates of 41.7% (5/12), whereas no queen (0/12) became pregnant by IVI (P=0.0083). In conclusion, an acceptable pregnancy rate was obtained with frozen-thawed semen in the domestic cat by non-surgical transcervical IUI; this method might also be useful in other small felids.

Sperm morphology in the domestic cat, and its relation with fertility: a retrospective study.

Knowledge about normal ranges in semen quality and the association between sperm morphology and fertility in felids is limited. The aims of this retrospective study were to (1) define a normal spermiogram in cats; (2) evaluate possible effects of season, age and breed on sperm morphology; and (3) evaluate the relationship between sperm morphology and fertility. Semen samples collected by electroejaculation from 52 cats were evaluated for sperm morphology. The cats constituted two groups: a general population of cats (n = 48) and cats examined because of poor breeding records (n = 4). The general population was divided into household (n = 20), pedigree (n = 19) and colony cats (n = 9) and into three age classes, <12 months, 12-59 months and >or=60 months. The median percentage of normal spermatozoa in the general population was 44.0% (range 1.0-91.0%). Criteria were tentatively set for what was considered a normal spermiogram. The mean percentage of normal spermatozoa was higher during February to July than during August to January (p < 0.05). Pedigree cats had a lower mean percentage of normal spermatozoa than did household cats (p < 0.05). Age had no effect on the percentage of normal spermatozoa but was positively correlated with the percentage of proximal droplets. Of the cats with <40% normal spermatozoa (n = 19), all those with known breeding records (n = 11) had produced litters. The four cats examined because of poor breeding results had higher percentages of different sperm abnormalities than tentatively stipulated for the normal spermiogram. In two of these cats both sperm morphology and fertility changed over time.

Epididymal and ejaculated cat spermatozoa are resistant to cold shock but egg yolk promotes sperm longevity during cold storage at 4 degrees C.

The aims were to evaluate the susceptibility of feline ejaculated and epididymal spermatozoa to cold shock and to evaluate the effect of egg yolk in the preservation extender. Ejaculated and epididymal spermatozoa from eight males were subjected to a slow (0.5 degrees C/min) or a fast (3 degrees C/min) cooling rate with controls kept in room temperature. Ejaculated and epididymal spermatozoa from another eight males were cooled in a plain Tris buffer (Tris) or in Tris with 20% egg yolk (EYT) and evaluated for 96 h. Subjective motility (MOT), plasma membrane integrity (PMI), and acrosome integrity (ACRI) were evaluated. Cooling did not induce sperm damage regarding PMI (P=0.6) or ACRI (P=0.19) and chilled spermatozoa had better overall MOT (P=0.046) than controls. EYT was better for MOT (P>0.05) from 48 h of cold storage than Tris. EYT was also better for overall ACRI (P<0.0001) while Tris was better for overall PMI (P=0.0004). There were no interactions between time and treatment (P>0.05) for PMI or ACRI. Ejaculated spermatozoa had better overall MOT (P<0.05) and PMI (P<0.05) than epididymal spermatozoa, and higher ACRI in experiment 1 (P=0.0003) but not in experiment 2 (P=0.117). Source of spermatozoa did not affect the susceptibility to cooling or the effect of egg yolk as there were no interactions (P>0.05) between source of spermatozoa and treatment (cooling or control) or between time, source and extender (P>0.05). In conclusion cat spermatozoa were tolerant to cold shock and egg yolk was beneficial for preservation of MOT and ACRI but not PMI.

Investigation of cervical patency and uterine appearance in domestic cats by fluoroscopy and scintigraphy.

The cervical patency of six domestic female cats was monitored under sedation by infusion of contrast medium (Omnipaque) into the cranial vagina during early oestrus, mid-oestrus, late oestrus and interoestrus or a radiopharmaceutical ((99m)Tc-HSA) during mid- and interoestrus in a non-ovulatory oestrous cycle. The transport of the contrast medium or the radiopharmaceutical through the cervix and within the uterine horns was observed under fluoroscopy and with the aid of scintigraphy. In three of the queens, transcervical transport of contrast medium was demonstrated in all stages of oestrus, in one queen during mid-oestrus, late oestrus and 1 day after oestrus, and in two queens only during late oestrus. The relations between the cervical patency to the contrast medium and the oestrous behaviour, cornification of the vaginal cells and the serum oestradiol-17beta concentration were evaluated, and a relationship was found between the cervical patency and the degree of vaginal cornification. Transcervical transport of the radiopharmaceutical was observed in three queens during mid-oestrus. When the cervix was open, hysterography under a fluoroscope and hysteroscintigraphy were performed. The fluoroscopic and scintigraphic recordings revealed the patterns of the uterine contractions during oestrus in both ascending and descending directions, and the movement of the uterine contents back and forth between the uterine horns. The hysterograms were classified according to the shape of the uterine horns and the appearance of the endometrial lining. Spiral-shaped uterine horns with a smooth inner contour were observed in two queens, and a corkscrew appearance with irregular filling defects in the uterine lumen was shown in two queens that had developed subclinical cystic endometrial hyperplasia. These findings demonstrated that fluids or particles deposited in the cranial vagina of the cat can be transported into the uterus during some stages of the oestrous cycle. The fluoroscopic and scintigraphic techniques developed in this study may be further modified to permit more detailed studies of uterine contractile patterns and sperm transport in the feline female reproductive tract. Hysterography proved useful to diagnose uterine disease. The information on cervical patency is of value also for the development of techniques for artificial insemination in this species, and should be studied also in the ovulatory cycle.