Single layer centrifugation as a method for bacterial reduction in bull semen for assisted reproduction.

Semen samples contain bacteria originating from the animal urogenital tract, environment, and/or contamination during semen processing, negatively affecting sperm quality by producing toxins and/or competing for nutrients in extenders. The aims of this study were to evaluate two methods of Single-layer centrifuges (SLC), high and low density colloid, as a method for bacterial removal from bull semen, and to evaluate sperm quality after treatment. In total, semen samples from 20 bulls (3 ejaculates per bull) were used in this study. Bacterial reduction was evaluated by bacterial quantification (colony forming unit - CFU/mL) while bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after culturing bacteria on blood agar. Sperm motility parameters were evaluated by Computer Assisted Sperm Analyses (CASA), and sperm chromatin structure assay (SCSA) by Flow cytometry. Both, High and Low density SLC reduced number of bacteria significantly (p < 0.001) compared with control. The difference in bacterial count between High and Low SLC was also significant (p < 0.001). Furthermore, High density SLC was successful in removing almost all Bacillus and Proteus spp. Most CASA parameters were significantly improved after both treatments (p < 0.001, p < 0.01, p < 0.05). The Deoxyribonucleic acid (DNA) fragmentation index evaluated by SCSA in High (p < 0.01) and Low (p < 0.05) SLC group differed significantly compared with control. Single-layer centrifugation (SLC) with either a high or a low density colloid is a suitable method for bacterial removal in bull semen.

Evaluation of Canine Neonatal Health by Breeders: A Prospective Questionnaire Study on the Association between Neonatal Scores (Modified APGAR), Parturition, Birth Weight, Growth, and Puppy Mortality.

Mortality of neonatal puppies is a widespread problem in small animal medicine. Neonatal monitoring, according to standardized protocols, can be useful for identifying puppies that are at risk of mortality. Prompt intervention on weak puppies could increase survival rates. Apgar scoring adapted for puppies has been demonstrated to be associated with mortality and is usually performed by trained veterinary staff. The majority of puppies, however, are born in a home or kennel environment and not at a veterinary clinic. Our aims were, therefore, to evaluate if a modified protocol for neonatal monitoring would be usable by breeders in a home environment. We wanted to evaluate potential associations between modified Apgar scores, birth weights, delivery times, growth rates, and puppy mortality. Modified Apgar scores were related to the viability of live-born puppies (p < 0.0013). The viability and expulsion time of each puppy were significantly related (p = 0.010 with all puppies included and p = 0.038 with only live-born puppies included). Viability was significantly related to relative birthweight (p < 0.01). Puppies with a negative growth rate the first two days after parturition did not have a significantly higher risk of mortality. In conclusion, a modified and simplified Apgar scoring performed by breeders approximately 5 min after birth was associated with puppy mortality.

Characteristics of reproductive organs and reproductive potential in Scandinavian female grey wolves (Canis lupus).

The Swedish wolf population is closely monitored and managed to keep the population at a sustainable level while avoiding conflicts. Detailed knowledge about reproduction is crucial for estimates of population size and the reproductive potential of a population. Post-mortem evaluation of reproductive organs can be used as a complementary tool to field monitoring for evaluation of cyclicity and previous pregnancy, including litter size. Therefore, we evaluated reproductive organs from 154 female wolves that were necropsied during the period 2007-2018. The reproductive organs were weighed, measured, and inspected according to a standardised protocol. Presence of placental scars was evaluated for estimates of previous pregnancy and litter size. Data about individual wolves were also obtained from national carnivore databases. Body weight increased during the first year of life before levelling out. There was evidence of cyclicity the first season after birth in 16.3 % of the 1-year-old females. No females < 2 years had evidence of a previous pregnancy. Pregnancy rates were significantly lower in 2- and 3-year old females than in older females. Mean uterine litter size was 4.9 ± 2.3, and did not differ significantly between age groups. Our data supports earlier field data that female wolves usually start to reproduce at the earliest at 2-years of age but that they occasionally start to cycle one season earlier. All females ≥ 4 years of age had reproduced. Pathological findings of the reproductive organs were rare, indicating that reproductive health of female wolves is not a limiting factor for population growth.

A Pilot Study to Evaluate the Potential of Melatonin Implants to Control Cyclicity in the Bitch.

In short-day breeders such as the sheep, melatonin stimulates oestrus activity; in contrast, a high serum concentration of melatonin inhibits oestrus in long-day breeders such as the cat. Therefore, implants with melatonin have been used to suppress or induce oestrus depending on the species. The aim of this pilot study was to evaluate if melatonin could be an alternative to control the reproductive cycle in the bitch. Nine beagle bitches were observed for three oestrus cycles. Five beagle bitches were treated with 18 mg melatonin implants on average 27 days before the next expected oestrus based on the previous interoestrus interval. Four bitches served as untreated controls. Blood samples for evaluation of serum melatonin were collected at the time of assignment to treatment or control group and 1-4 weeks thereafter. Clinical signs and vaginal smears were used to follow the cycle. Melatonin varied significantly with bitch (p < 0.05) but not with treatment. Treatment did not affect the interoestrus interval (p > 0.05). In conclusion, treatment with 18 mg melatonin implants approximately one month before expected oestrus is not likely to be a useful method to control cyclicity in the bitch. It is still not known if melatonin is involved in regulation of the oestrus cycle in the domestic dog.

Factors affecting reproductive performance in the Swedish Bernese mountain dog.

BACKGROUND: Good reproductive performance is fundamental for the development of a breed. Previous studies have indicated that the Bernese mountain dog has a relatively high prevalence of reproductive problems such as a high prevalence of dystocia and a low mean litter size. When reproduction is impaired, selection for other traits, including improved health, will become more difficult. The aim of this study was therefore to evaluate reproductive data and factors affecting these in the Bernese mountain dog. Data collected by the Swedish Sennenhund Club during the years 2010-2020 were evaluated by statistical analyses. RESULTS: Information from 1287 reported matings were included with a total of 614 bitches and 399 sires. For five reported matings that did not result in a litter, there was no information about the male identity. The reported matings resulted in 798 litters (62% whelping rate) from 502 bitches and 314 males. Paternal and maternal age had a significant effect on whelping rate with a negative effect of increasing age (P < 0.01). Median litter size at birth (LSB) was 6.00 (range 0-14) and was significantly affected by both paternal (P = 0.021) and maternal age (P < 0.001). Parity affected litter size at birth with a lower litter size in 4 year old bitches giving birth to their first litter compared to bitches giving birth to their second to fourth litters. Stillbirth occurred in 51.6% of the litters with a total of 15.4% puppies being stillborn. Total puppy mortality, including stillbirth, was 19.1%. The only factor affecting stillbirth was LSB while both LSB and season affected the risk of having post-natal puppy loss in the litter. The total prevalence of caesarean sections (CS) was 33.0%. The risk of CS decreased significantly with increasing parity and increased with increasing age. The risk of CS was significantly higher for litters with 1-2 puppies compared with litters with 3-9 puppies. The coefficient of inbreeding (F) calculated on 5 generations had no effect on any of the outcomes. CONCLUSIONS: Parity and maternal age had opposite effects on reproductive outcomes with a positive effect of parity on increasing litter size and decreasing CS rate. The proportion of unsuccessful matings was high with a negative effect of increasing age of both males and females.

Cryopreservation of dog semen in a Tris extender with two different 1% soybean preparations compared with a Tris egg yolk extender.

Egg yolk is widely used as a cryoprotectant in dog semen extenders, but there is a risk of contamination with animal pathogens. In addition, egg yolk may vary in composition, making it difficult to standardize the extender. Lecithin is an animal protein-free alternative to egg yolk for semen cryopreservation. Recently, it was shown that 1% of soybean lecithin type II-S was better than 2% for freezing canine semen. The aim of the study was to compare two different types of soybean lecithin, with egg yolk as a control. Ejaculates from eight dogs were divided into three equal parts and diluted with a Tris-based extender, containing either 20% egg yolk, 1% soybean lecithin Type II-S or 1% soybean lecithin Type IV-S. The samples were then frozen. Sperm motility was evaluated by computer-assisted sperm analysis (CASA), acrosome integrity (FITC-PNA/PI) and sperm membrane integrity (SYBR-14/PI) post-thaw, as well as after 2 and 4 hr incubation at 37°C. Post-thaw sperm chromatin structure assay and plasma membrane integrity were evaluated by flow cytometry. Total motility, sperm plasma membrane integrity and acrosome integrity were significantly better in the egg yolk extender than in the two soybean lecithin-based extenders. Individual motility post-thaw differed more than in the fresh samples, illustrating individual differences in tolerance to the cryostress. The DNA Fragmentation Index (% DFI) was significantly lower in the Tris egg yolk (TEY) extender compared to any of the soybean-based extenders. The number of high green stained spermatozoa were significantly higher in Type IV-S compared to the control TEY extender. In conclusion, egg yolk was superior to the two lecithin-based extenders to cryopreserve canine semen.

Characteristics of reproductive organs and estimates of reproductive potential in Scandinavian male grey wolves (Canis lupus).

Improved knowledge about reproductive patterns and potential in male wolves (i.e., testicular development and size relative to age, pubertal age, and seasonal effects) is needed for evaluation and monitoring of reproductive outcomes in populations. Reproductive organs from 215 male wolves, culled as a result of licensed hunting, protective culling or from carcasses found were examined. The testes and epididymis were weighed and measured. There were biopsy samples collected from the testes and the cauda epididymis for histological determinations if there were spermatozoa in tissues collected. There were reproductive tissue analyses of 197 males while there were separate evaluations of tissues from ten cryptorchid animals. Juvenile wolves (< 1 year, n = 47) had a lesser body mass and mean testes mass than subadult (1-2 years, n = 71) and adult (>2 years, n = 79) males. Season also affected testicular characteristics of structures evaluated with subadult and adult males having a lesser mass during summer months (May-August). Of the 197 males, 70 % had spermatozoa in the seminiferous tubules and the cauda epididymis and were classified as being 'potentially fertile' when tissues were collected, while 22 % were classified as being non-fertile (no spermatozoa, including males that were pre-pubertal) and tissues of 8% could not be evaluated. When testes mass was greater, there was a greater likelihood that spermatozoa were present. There were seven of the ten cryptorchid males of the unilateral type. These testicular and epididymal findings will be useful for evaluating the reproductive potential and management of wolves in Scandinavia.

Extenders for alpaca epididymal spermatozoa: Comparison of INRA96 and andromed.

Artificial insemination would be a useful technique for alpaca breeders to use as an aid to breeding to increase fleece quality. The technique, however, is not well developed in alpacas, partly because of the viscous nature of their seminal plasma. Castration conducted for husbandry purposes can provide a source of epididymal spermatozoa to test semen extenders or handling regimens, thus circumventing the problem of the viscous ejaculate. In this experiment, two semen extenders (Andromed and INRA96) developed for other species (bovine and equine, respectively) were tested with alpaca spermatozoa derived from the cauda epididymis. Sperm total motility (mean ± SEM A: 29.1 ± 4.8 % compared with I: 35.4 ± 4.8 %; NS), membrane integrity (A: 58 ± 9% compared with I: 56 ± 9%; NS) and acrosome integrity (A: 65 ± 7% compared with I: 54 ± 7%; NS) were not different between the two extenders. Progressive motility with use of INRA96 was greater after incubating for 30 min than after incubating for 10 min (35 ± 4% vs. 12 ± 4%, respectively; P = 0.03). In conclusion, viable epididymal spermatozoa could be extracted from the castrated organs after overnight transport. There were no differences in sperm quality between the two extenders; therefore, it appears that either extender could be used for alpaca spermatozoa. These results could help in the development of a technique for artificial insemination in alpacas.

Inflammatory changes during canine pregnancy.

Pregnancy is considered a pro-inflammatory state that requires physiologic adaptation of the immune system of the mother. The aim of the present study was to study inflammatory and hormonal changes during canine pregnancy. Studies included analyses of peripheral concentrations of the acute phase proteins fibrinogen and C-reactive protein (CRP), the hormones progesterone and insulin-like growth factor I (IGF-I), hemoglobin, and analyses of the total leukocyte numbers and expression of cell surface antigens. Twenty bitches were included in the present study; 12 pregnant bitches and eight non-pregnant control bitches that were followed during the corresponding phase of the oestrous cycle. Blood samples were collected at the day of optimal mating (day 0) and then on days 7, 14, 21, 28 and 42. Progesterone, IGF-I and CRP were analysed in serum and fibrinogen in EDTA plasma. Haematology and leukocyte expression of a panel of inflammation-associated adhesion molecules (CD 11a, CD 18 and CD 49d) were evaluated from EDTA blood. The data were analyzed as repeated-measures data, using a mixed model approach. Progesterone varied with time in both pregnant and control bitches, and IGF-I varied with time in pregnant bitches. Both fibrinogen and CRP increased significantly with time for the pregnant bitches, but no significant change was detected for the control bitches. Increases were seen from day 21. The hemoglobin concentration decreased significantly with time in both pregnant and non-pregnant bitches. The neutrophil and monocyte numbers increased significantly in pregnant but not in control bitches. Pregnancy induced increased granulocyte expression of cell surface marker CD 18, increased monocyte expression of CD 18 and CD 49d, and increased lymphocyte expression of CD 49d. In conclusion, we describe inflammatory changes during canine pregnancy that are manifested as increases in concentrations of CRP and fibrinogen, an increase in neutrophils and monocytes, and in activation of granulocytes, monocytes and lymphocytes. The changes should be taken into account when evaluating concentrations of APPs and WBC in bitches during pregnancy. A variation in IGF-I concentrations was detected during pregnancy.

Osmotic tolerance of feline epididymal spermatozoa.

During the cryopreservation process, spermatozoa are exposed to hypertonic solutions contributed by the high concentration of cryoprotectant. During addition and removal of cryoprotectant the spermatozoa are subjected to a substantial osmotic stress. Spermatozoa of different species and different stages of maturation may have different susceptibility to osmotic stress depending on the biology of the cell membrane and this will affect their tolerance to the freezing-thawing stress. The aims of this study were to determine the osmotic tolerance limits for motility, membrane integrity and mitochondrial membrane potential of feline epididymal spermatozoa and to study the effect of osmotic stress on the feline spermatozoa of different epididymal regions. Epididymal spermatozoa from three regions (caput, corpus and cauda) were pre-exposed to various osmolalities (75, 300, 600, 900, 1200 mOsm) in a single step for 10min and returned to 300 mOsm afterward. Percentage of motile spermatozoa was measured subjectively and membrane integrity (SYBR-14 positive cells) was evaluated prior to and after exposure to different osmolalities. The mitochondrial membrane potential (JC1) of spermatozoa were evaluated using flow cytometer and compared between epididymal regions (caput, corpus and cauda). All the parameters were compared using a mixed procedure. The percentage of motile epididymal spermatozoa decreased significantly when spermatozoa were exposed to 75 mOsm and 600 mOsm. Epididymal spermatozoa showed signs of damage when pre-exposed to 900 and 1200 mOsm and returned to isotonic condition as significant reduction of membrane integrity and mitochondrial membrane potential were observed (P<0.05). The plasma membrane of spermatozoa from the cauda epididymal region showed higher susceptibility to osmotic stress than the other regions as demonstrated by a significant difference between regions after return to isotonicity from 900 mOsm (P>0.01) and a difference between caput and corpus after return from 1200 mOsm (P<0.05). The corpus and cauda epididymal spermatozoa had higher percentage of spermatozoa with high mitochondrial membrane potential than those from caput when exposed to 75, 300 and 600 mOsm (P<0.05). In conclusion, a single step exposure to hypertonic solution of greater than 600 mOsm prior to return to isotonic condition can cause severe damage to sperm membrane and mitochondrial membrane potential compared to non-returning (exposure to various osmolality but not returned to isotonic condition). Changes in osmolality impacted mostly on sperm motility. Spermatozoa from cauda epididymis were more susceptible to osmotic stress compared to those from corpus and caput indicating that the maturation changes in the sperm membrane during passage through the epididymis increase susceptibility to the osmotic changes that may occur during sperm cryopreservation.

Dystocia in the cat evaluated using an insurance database.

Objectives The aim of this study was to describe the incidence of feline dystocia with respect to breed. Methods The data used were reimbursed claims for veterinary care insurance and/or life insurance claims in cats registered in a Swedish insurance database from 1999-2006. Results The incidence rates for dystocia were about 22 cats per 10,000 cat-years at risk, 67 per 10,000 for purebred cats and seven per 10,000 for domestic shorthair cats. The median age was 2.5 years. A significant effect of breed was seen. An incidence rate ratio (IRR) that was significantly higher compared with other purebred cats was seen in the British Shorthair (IRR 2.5), the Oriental group (IRR 2.2), Birman (IRR 1.7), Ragdoll (IRR 1.5) and the Abyssinian group (IRR 1.5). A significantly lower IRR was seen in the Norwegian Forest Cat (IRR 0.38), the Maine Coon (IRR 0.48), the Persian/Exotic group (IRR 0.49) and the Cornish Rex (IRR 0.50). No common factor among the high-risk breeds explained their high risk for dystocia. There was no effect of location; that is, the incidence rate did not differ depending on whether the cat lived in an urban or rural area. Caesarean section was performed in 56% of the cats with dystocia, and the case fatality was 2%. Conclusions and relevance The incidence rate for dystocia was of a similar magnitude in purebred cats as in dogs. The IRR varied significantly among breeds, and the main cause for dystocia should be identified separately for each breed. A selection for easy parturitions in breeding programmes is suggested.

In vitro fertilization using frozen-thawed feline epididymal spermatozoa from corpus and cauda regions.

Epididymal sperm preservation offers a potential for rescuing genetic material from endangered or valuable animals after injury or death. Spermatozoa from corpus, as well as from cauda, have the capability to be motile and to undergo capacitation and can thus potentially be preserved for assisted reproductive technologies. In the present study, feline frozen-thawed epididymal spermatozoa from corpus and cauda regions were investigated for their ability to fertilize homologous oocytes and further embryo development in vitro. Epididymal spermatozoa from corpus and cauda of seven cats were cryopreserved and used for IVF. Cumulus-oocyte complexes (n = 419) were obtained from female cats after routine spaying. Frozen-thawed corpus epididymal spermatozoa showed similar properties of acrosome integrity, membrane integrity, and chromatin integrity as frozen-thawed spermatozoa from cauda except corpus spermatozoa showed lower motility (P < 0.05). The fertilizing capacity of frozen-thawed corpus epididymal spermatozoa was confirmed by similar number of embryos developing to the two- and four-cell stages compared with sperm from cauda (32.03% vs. 33.33%). However, oocytes fertilized with corpus spermatozoa had lower potential to develop to the blastocyst stage (6.79%) and had lower cell numbers compared to oocytes fertilized with cauda spermatozoa (14.08%). In conclusion, spermatozoa from corpus epididymis had a similar capability to fertilize homologous oocytes in vitro as sperm from cauda but resulted in fewer embryos developing to the blastocyst stage compared to spermatozoa from the cauda.

The tolerance of feline corpus and cauda spermatozoa to cryostress.

Epididymal sperm preservation can be used to avoid the total loss of genetic material in threatened species. Spermatozoa from the corpus, as from the cauda, are motile and can undergo capacitation. Thus, they can potentially be preserved for assisted reproductive technologies. However, cryopreservation of spermatozoa has a direct detrimental effect on sperm quality. The aim of this study was to compare the chromatin stability and the survival rate of spermatozoa from the corpus and cauda epididymis after cryopreservation. Epididymal spermatozoa were collected and cryopreserved from the corpus and cauda of 12 domestic cats. Sperm motility, progressive motility, membrane integrity, acrosome integrity, and DNA integrity were evaluated before and after freezing thawing. The average total number of spermatozoa collected from the corpus was lower (10.2 × 10(6) ± 7.4) than that from the cauda epididymis (24.9 × 10(6) ± 14.4; P = 0.005). The percentage of spermatozoa with intact DNA did not differ significantly whether it was collected from the corpus or cauda regions and did not decrease after freezing thawing in either region. However, motility of spermatozoa from both regions was affected by the freezing thawing process with a significant decline in motility after thaw compared with fresh spermatozoa. A significant difference in the percentage of motile sperm between the corpus and cauda was observed after the freezing thawing process (P < 0.001). Although sperm motility was lower in postthaw spermatozoa from the corpus epididymidis than from the cauda, the rate of the reduction did not differ between regions. This study indicates that the cryopreservation process does not have a negative effect on chromatin stability of feline epididymal spermatozoa. Spermatozoa from the corpus region have a similar freezability as spermatozoa from the cauda region. Therefore, preservation of spermatozoa from the corpus and the cauda epididymidis might be of value in preserving genetic material from endangered or valuable felids.

The ability of feline spermatozoa in different epididymal regions to undergo capacitation and acrosome reaction.

The sperm maturation process that occurs in the epididymis is a necessary process for spermatozoa to acquire motility and the ability to undergo capacitation, which is an important key for fertilization. The aim of this study was to evaluate the ability of feline spermatozoa from different regions of the epididymis to undergo capacitation and acrosome reaction. Experiment I: epididymal spermatozoa from caput, corpus and cauda regions were placed in phosphate buffered saline (control medium) and in vitro fertilization medium (capacitating conditions). Sperm motility, motility patterns, plasma membrane integrity and tyrosine phosphorylation were evaluated at time 0 and 60min after incubation. Experiment II: spermatozoa were treated with 2µM of calcium ionophore (A23187) to induce the acrosome reaction and acrosome reaction was evaluated. The results showed a significant effect of region with a higher percentage of tyrosine phosphorylation in spermatozoa from the cauda than in the caput or corpus regions (P=0.0061; P=0.0088). Spermatozoa from corpus and cauda showed higher values in the majority of the measured motility parameters than spermatozoa from the caput (P<0.0001). Spermatozoa from all epididymal regions can undergo the acrosome reaction in vitro in response to induction by calcium ionophore with no difference between regions (P>0.05). Spermatozoa from all epididymal regions were able to undergo capacitation. Higher percentage of tyrosine phosphorylation in spermatozoa from the cauda reflect that they more easily underwent capacitation compared to spermatozoa from caput and corpus which required more time of incubation for capacitation. In conclusion feline epididymal spermatozoa from all regions can undergo capacitation and acrosome reaction in vitro and do not require incubation under capacitating conditions.

A questionnaire on survival of kittens depending on the blood groups of the parents.

Cats more than 2 months of age have alloantibodies against the blood type antigen that they do not possess. Maternal antibodies, including alloantibodies against blood groups, are transferred to the kittens' systemic circulation when they suckle colostrum during the first 12-16 h after birth. If kittens with blood group A or AB nurse from a mother with blood group B they may develop neonatal isoerythrolysis (NI). Breeders can prevent kittens at risk of NI from nursing during the first 16-24 h; after this period it is safe to let them nurse. Kittens depend, however, on the passive transfer of antibodies from the colostrum for early protection against infections. Although it is known that kittens deprived of colostrum will also be deprived of passive systemic immunity, it is not known if this will affect their health. Therefore, the aim of this study was to evaluate kitten mortality in litters with B-mothers and A-fathers compared to litters with A-mothers. In addition, the aim was to evaluate the effects of colostrum deprivation on the health of the mothers, and the breeders' opinions and experiences of these combinations of breedings. A web-based questionnaire was constructed and distributed to breeders. The results indicate that there is no difference in mortality between planned litters that have mothers with blood group A and litters with mothers that have blood group B and fathers that have blood group A. When managing blood group incompatibility in cat all factors affecting the health of the cats, including genetic variation, should be considered.

An internet survey of breeders' and cat rescue organisations' opinions about early castration of cats.

There has been concern that early castration of pedigree kittens may lead to a depletion of gene pools. Web-based questionnaires on early castration were distributed to breeders and cat rescue organisations. One of the reasons that breeders used early castration was to counteract what they considered irresponsible breeding, such as overuse of strains within the breed or production of cross-breeds. Of all pedigree kittens, 45.1% were kept intact while 54.9% were neutered before re-homing. Nineteen (65.5%) of the cat rescue organisations believed that early castration could be beneficial in reducing the number of homeless cats, but only six (20.6%) had applied early castration. Three organisations replied that their veterinarian declined to do early castration and two believed that it was not safe for the kittens. There does not, necessarily, seem to be conflicting interests between keeping genetic variation in pedigree breeds and the possibility of limiting the population of homeless cats.

Infertility in the cycling queen: seven cases.

In this study we examined seven queens with normal oestrous cycles and a history of infertility after normal matings. We performed clinical examination, vaginal cytology, evaluation of oestradiol, progesterone and total T4 levels, vaginal bacterial culture, ultrasonography, and serum analyses for detection of antibodies against chlamydia and feline leukaemia virus (FeLV) antigenemia. Ovariohysterectomy (OHE) was recommended for 1/7 queens because of pathological uterine changes detected at ultrasonography and clinical examination. Four out of seven queens were treated with antibiotics and two of these had more litters. One of the queens that were treated was not mated again and one was mated without conceiving and was at a later OHE found to have degenerative uterine changes. No treatment was given and no diagnosis could be established in 2/7 queens. Both of them were later ovariohysterectomised and one showed degenerative uterine changes while the uterus of the other queen could not be obtained for follow-up. In summary, 4/7 cats were diagnosed with uterine pathology and no definitive diagnosis could be established for 3/7 queens.

Application of a zona pellucida binding assay (ZBA) in the domestic cat benefits from the use of in vitro matured oocytes.

BACKGROUND: Zona pellucida binding assays (ZBAs) have proven useful in determining the fertilising ability of spermatozoa in several species. Most ZBAs use fresh or salt-stored oocytes collected from fresh ovaries but because ovaries are not easy to obtain on a regular basis, chilled and frozen-thawed ovaries have been tested, with varying results. The present study tested the hypothesis that cat spermatozoa, either fresh or frozen-thawed, can bind to homologous zona pellucida of oocytes retrieved from frozen-thawed queen ovaries to a similar extent as they can bind to the zona pellucida of fresh, in vitro matured oocytes. METHODS: Ovaries were collected from queens after routine ovario-hysterectomy and either stored in NaCl at -20 degrees C until use (treatment animals), or used fresh (controls). Cumulus-oocyte complexes (COCs) were retrieved by ovarian slicing from either source and used directly (immature oocytes from frozen-thawed ovaries; treatment animals) or after in vitro maturation (IVM) (fresh ovaries; controls) for 24 hours in TCM 199, supplemented with 1 IU hCG/mL and 0.5 IU eCG/mL and 0.5% bovine serum albumin (BSA). The oocytes were incubated for 4 hours in 5% CO2 in air at 38 degrees C and 100% humidity in the presence of 5 x 106 fresh or frozen-thawed spermatozoa/mL. Representative samples of oocytes were processed for scanning electron microscopy (SEM). RESULTS: Both fresh and frozen-thawed spermatozoa bound to the in vitro matured zona pellucida but significantly fewer, or no, spermatozoa bound to frozen-thawed, immature zona pellucida (P < 0.001). Also, more fresh spermatozoa than frozen-thawed spermatozoa bound to the zona pellucida (P < 0.001). The zona pellucida surface differed in morphology (SEM), with in vitro matured oocytes showing a dense surface with few fenestrations in contrast to their frozen-thawed, immature counterparts, where fenestrations were conspicuously larger. CONCLUSION: In conclusion, under the conditions of the present study, immature oocytes recovered from ovaries frozen immersed in NaCl at -20 degrees C are less suitable for use in feline ZBA.

Sperm maturation in the domestic cat.

The epididymis is essential for sperm development and maturation, and, subsequently, the ability of spermatozoa to penetrate and fertilize the female gamete. Functional differences in segments of the long tubule are reflected by histological differences among epididymal regions. The feline epididymis can be divided into six different regions according to their histological differences. A marked increase in sperm concentration occurs between regions 2 and 3, indicating resorption of fluid in region 2, a concept supported by the histological characteristics of the epithelium. At the transition between regions 4 and 5, located between the caput and corpus epididymides, histological characteristics change from being that of a maturation function to being typical of a storage function. Migration of the cytoplasmic droplet and induction of motility occur in this same region. Proteins are secreted from epithelial cells in the feline epididymis by merocrine and apocrine secretion, although the functions of different feline epididymal proteins have not been determined. Hypotaurine, taurine and, probably, alkaline phosphatase are produced by the feline epididymis. During epididymal transit the percentage of immature, unviable and morphologically abnormal spermatozoa decreases, indicating the existence of a mechanism that removes abnormal spermatozoa. In contrast, the percentage of spermatozoa with abnormal tails increases slightly during epididymal transit. Most of the distal droplets present on spermatozoa in the cauda epididymis are lost at or after ejaculation. Additional knowledge of the feline epididymis should be beneficial for developing sperm preservation protocols and advance the prospects for effective male contraceptive methods.

Cervical patency during non-ovulatory and ovulatory estrus cycles in domestic cats.

The cervix functions as a barrier to spermatozoa. Vaginal artificial insemination in cats is, therefore, likely to be successful only at the period of estrus when the cervix is open. This study aimed to define the period of cervical patency in cats in both non-ovulatory and ovulatory estrus cycles. A total of 15 reproductive cycles were studied in six cats during the estrous stage. Cervical patency was monitored with the cats under sedation, by infusing 2 mL of Iohexol contrast medium via a 3.5 French tomcat catheter into the cranial vagina during estrus. Day one of estrus was defined as the first day the cats showed estrous behavior. Non-ovulatory cycles were characterized by a serum progesterone concentration on days 11-15 that was below 5 nmol/L and a normal interestrus interval of 7-14 days. Ovulatory cycles were characterized by a serum progesterone concentration on days 11-15 that was above 5 nmol/L and an interestrus interval that exceeded 30 days. The cervix was considered to be open when the contrast medium was seen to enter the uterus, and to be closed when the contrast medium remained in the vagina. Blood samples were collected at each examination and were assayed for estradiol-17beta and progesterone concentrations. The cervix was open on the first day of standing estrus at a mean estradiol-17beta serum concentration of 87.4+/-21.8 pmol/L (range 14 to >or=180 pmol/L) and closed at an estradiol concentration of 47.1+/-12.4 pmol/L (range 4 to >or=180 pmol/L). In the ovulatory cycles the cervix was closed at a progesterone concentration of 9.8+/-4.4 nmol/L (range 0.6-28.4 nmol/L). There was no difference in the duration of cervical patency in non-ovulatory and ovulatory cycles (5.5+/-1.2 days and 5.2+/-0.5 days, respectively) (p>0.05). The higher overall mean concentrations of estradiol-17beta seen in the ovulatory cycles than in the non-ovulatory cycles, indicate that a high level of estradiol is necessary for induction of ovulation. Ovulation in 60% of unmated females in this study indicates that the techniques used for evaluation of cyclus stage and cervical opening have the potential to induce ovulation in the cat. This study demonstrates that cervical patency is not influenced by the occurrence of ovulation, but is due to individual variations between cats.