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Sex identification in mammalian preimplantation embryos is a technique that is used currently for development of the embryo transfer industry for zootechnical animals and is, therefore, a resource for biodiversity preservation. The aim of the present study was to establish a rapid and reliable method for the sexing of preimplantation embryos in domestic cats. Here we describe the use of nested PCR identify Y chromosome-linked markers when starting from small amounts of DNA and test the method for the purpose of sexing different species of wild felids. To evaluate the efficiency of the primers, PCR analysis were performed first in blood samples of sex-known domestic cats. Cat embryos were produced both in vitro and in vivo and the blastocysts were biopsied. A Magnetic Resin System was used to capture a consistent amount of DNA from embryo biopsy and wild felid hairs. The results from nested PCR applied on cat blood that corresponded to the phenotypical sex. Nested PCR was also applied to 37 embryo biopsies and the final result was: 21 males and 16 females. Furthermore, beta-actin was amplified in each sample, as a positive control for DNA presence. Subsequently, nested PCR was performed on blood and hair samples from some wild felines and again the genotyping results and phenotype sex corresponded. The data show that this method is a rapid and repeatable option for sex determination in domestic cat embryos and some wild felids and that a small amount of cells is sufficient to obtain a reliable result. This technique, therefore, affords investigators a new approach that they can insert in the safeguard programmes of felida biodiversity.

The leopard cat (Prionailurus bengalensis), a member of the felidae family, is a threatened animal in South Korea. In terms of protecting endangered felids, nuclear transfer (NT) is a potentially valuable technique for assuring the continuation of species with dwindling numbers. In the present experiment, nuclear and microtubule remodeling and the in vitro developmental potential of enucleated domestic cat oocytes reconstructed with nuclei of somatic cells from either domestic cat fibroblast (DCF) or leopard cat fibroblast (LCF) were evaluated. Microtubule aster is allocated to de-condensed chromatin following nuclear transfer (3h after activation) of fibroblast cells from both domestic and leopard cats, suggesting the introduction of a somatic cell centrosome. The transferred fibroblast nuclei formed a large, swollen, pronuclear-like structure in most reconstructed oocytes, in the cat or leopard cat. At 18h following nuclear transfer, mitosis occurred, and according to the photo (F) it appears that spindle microtubules and two asters were observed. The percentages of blastocyst formation from nuclear transfer embryos derived from domestic cat fibroblasts (4/46, 8.6%) were not significantly different than those for nuclear transfer embryos constructed with leopard cat fibroblasts (4/52, 7.6%). These results indicate that nuclear and microtubule remodeling processes and in vitro developmental ability are similar in reconstructed cat oocytes following transfer of nuclei from either domestic or leopard cats.

The aim of this research was to compare the morphological aspects during the development of pregnancy in dogs and cats, distinguishing features of the fetal membranes, such as yolk sac evolution and differentiation of hemangioblasts, and the degree of elaboration of the amnion and allantois. Canine and feline placentae from 20, 24, 35, 45 and 55 d of pregnancy were perfusion-fixed for histological investigation and vascular corrosion casts were produced. The casts were prepared for scanning electron microscopy (SEM) and the embryo and fetal membrane development was analyzed. The growth patterns of the conceptuses were compared with the organization of the placentation process, and changes of the morphology during pregnancy were recorded. In feline placentae, an incomplete zonary shape was present in 62.5% out of 60 studied cases. This was located distal to the insertion of the umbilical cord. In the lamellar zone, the interhemal membrane or placental barrier resembled endotheliochorial conditions, and the maternal-fetal microvascular blood flow interrelationship was of simple crosscurrent type. Dogs have a zonary placenta, completely surrounding the fetus, and complex lamellar organization of maternal and fetal tissues. At the border, two marginal hematomes with green colouration delimited the central placental girdle. The yolk sac consisted of one large sacculation with an inverted "T" shape and an enormous number of blood vessels; it had hemangioblast cells in contact with the epithelium. The amnion was avascular in early stages, but became vascularized by blood vessels of the internal allantoic membrane in later stages of pregnancy by intrinsic relation.

The objective was to assess the ability of testicular spermatozoa to fertilize in vitro matured domestic cat oocytes and support blastocyst formation in vitro following intra-cytoplasmic sperm injection (ICSI). After IVM, oocytes were randomly and equally allocated among treatment groups (ICSI with testicular spermatozoa, ICSI with ejaculated spermatozoa, sham ICSI, and control IVF). At 18 h after either injection or insemination, the percentage of fertilized oocytes (per total metaphase II oocytes) was approximately 65% after ICSI with testicular or ejaculated spermatozoa (P > 0.05), which was less (P < 0.05) than control IVF (approximately 90%). On Day 7, the percentage of cleaved embryos (per total metaphase II oocytes) was approximately 60% after ICSI with testicular or ejaculated spermatozoa (P > 0.05), which also was less (P < 0.05) than control IVF (approximately 85%). After ICSI with testicular spermatozoa, the percentage of blastocysts (per total cleaved embryos) was approximately 11.0%, which was less (P < 0.05) than ICSI with ejaculated spermatozoa (approximately 21.0%); the latter was less (P < 0.05) than control IVF (approximately 43.0%). No blastocyst formation was observed after sham ICSI. For the first time in the domestic cat, this study demonstrated the fertilizing ability and developmental potential of intra-testicular spermatozoa delivered directly into intra-ovarian oocytes matured in vitro.

The present study was conducted to determine the effect of electric field strength on the rate of membrane fusion between the somatic cell and cytoplast and on subsequent in vitro development of reconstructed embryos. Additionally, the in vitro developmental competence of cat oocytes artificially activated after 44 h of maturation culture was examined. An efficient fusion rate (64.2%) was obtained by applying a single pulse of 1.5 kV/cm for 50 micros, and the fusion rate remained almost constant at the higher field intensity (59.8 and 54.9% at 1.7 and 2.0 kV/cm, respectively). Although the cleavage rate of fused embryos increased with an increase of the electric field strength, there were no differences among the groups with respect to the proportion of development to the morula and blastocyst stages. In the additional experiment, oocytes at the metaphase II stage after culture for 44 h were activated by the combination of calcium ionophore (CaI) with cycloheximide (CHX). Some (11.8%) of activated oocytes developed to the blastocyst stage. Results from this study indicated that electric field strength affects the rates of fusion and cleavage but has no significant effects on the development to the blastocyst stage of reconstructed embryos. Prolonged maturation culture of cat oocytes (up to 44 h) decreased their ability to develop to the blastocyst stage.

The leopard cat (Prionailurus bengalensis), a member of the felidae family, is currently listed as threatened by the Ministry of Environment in South Korea. In exotic or endangered species, the lack of oocytes and recipients precludes the use of traditional somatic cell nuclear transfer, and an approach such as inter-genus nuclear transfer may be the only alternative for producing embryos and offspring. In the present study, we used the leopard cat as a somatic cell donor to evaluate the in vivo developmental competence, after transfer into domestic cat recipients, of cloned embryos produced by the fusion of leopard cat fibroblast cell nuclei with domestic cat cytoplasts. A total of 412 enucleated domestic cat oocytes were reconstructed with either male (Group A) or female (Group B) adult leopard cat fibroblasts. There was no significant difference in fusion rate (60.4% versus 56.9%) between Groups A and B. Of the cultured embryos, the cleavage and blastocyst developmental rate were not significantly different between Groups A and B (69.5% versus 60.8%; 7.2% versus 7.8%, P > 0.05). In Group A, in vivo developmental studies at 30-45 days postimplantation demonstrated 4.8% (21/435) of reconstructed embryos (n = 435) had entered into the uterine lining of recipients, while 1.4% (6/435) formed fetuses. However, all of the reconstructed embryos failed to develop to term (65 days). Microsatellite analyses confirmed that the nuclear genome of the cloned fetus were leopard cat in origin.

Domestic cat embryos of excellent quality appear to improve development of conspecific embryos when cultured together, providing an avenue for improving development of embryos from valuable species or individuals. To have relevance to rare species, it would be useful to understand if this advantage could be conferred by heterospecific companions because there usually are severely limited numbers of conspecific embryos available from wildlife donors. In the first study, we incubated single test cat embryos alone (controls) or with 10 cat embryos or 10 or 20 mouse embryos under similar regimented conditions (each group shared 20 microl medium). In the second study, single test cat embryos were cultured alone, with 10 conspecific or 20 mouse embryos or 10 cattle embryos (each group shared 20 microl medium). Single test embryos in all treatment groups achieved similar (P>0.05) stages of compaction and blastocyst development. In the first study, only the test embryos incubated with 10 cat or 20 mouse companion embryos achieved blastocyst expansion. The average total cell number within test embryos incubated with 10 cat or 20 mouse companions was greater (P<0.05) than controls or those placed with 10 mouse embryos. In the second study, test embryos in all groups achieved blastocyst expansion and had more (P<0.05) total cells per embryo than the solitary controls. Inner cell mass to trophoblast cell ratio did not differ among treatments in either study. Thus, companion mouse and cattle embryos selected for excellent quality confer a benefit to singleton cat embryos, although the number of companions necessary to grant an advantage may be species dependent. If this phenomenon can be extrapolated across species, this may be an avenue for 'common animal embryos' to improve developmental potential of embryos from rare, unrelated taxa.

Appreciable progress has been made in the development of assisted reproductive technology (ART) for creating in vitro embryos in cats. Moreover, the extent of advancement in the last decade has been similar, albeit of more modest magnitude, to that seen in some other domestic and laboratory species, particularly when the disparities in financial, and, hence, scientific, resources are considered. The recent progress in domestic felid ART has made it possible to envisage their potential role in supporting the conservation of endangered felid species, which, in reality, is a multifarious process requiring wide-ranging, yet coordinated approaches. The prospect of incorporating ART into that intricate domain, with limited exceptions, remains a long-term, but highly motivating objective. Meanwhile, the straightforward accessibility and abundant supply of domestic cat gametes from local veterinary clinics provides a valuable and practical source of material for further research on the basic aspects of in vitro oocyte maturation, fertilization and early embryo development. Furthermore, extrapolating the domestic biotechniques to non-domestic felids has produced encouraging results in some species.

The dynamic regulation of mammalian folliculogenesis is a key component of the reproductive process. Traditionally, the rodent had been used as a model to study ovarian function and reproductive physiology due to the availability of animals, their relatively short cycle length, high rate of fecundity and short generation interval. We maintain that much basic information can be determined using domestic cat ovaries retrieved from local veterinary clinics following routine spaying, without having the expense of maintaining a colony of laboratory cats. Studies of normal feline reproductive physiology and advances in reproductive technology may be extrapolated for use in endangered non-domestic felids. Increased understanding of feline reproduction will be beneficial to veterinary medicine, and to groups working to control feral cat populations. It is important to examine reproductive mechanisms in alternative animal models as there are a vast number of threatened and endangered species in which we lack the critical reproductive information needed to assist in preserving their long-term survival.

The objective of the study was to investigate interspecies Somatic Cell Nuclear Transfer (iSCNT) techniques in marbled cats (Pardofelis marmorata), using domestic cat and rabbit oocytes as the recipient cytoplasm. The recipient oocytes were obtained from ovariohysterectomized cats and superovulated rabbits. The donor cells were collected from a male marbled cat that had died in captivity. Experiment 1 was conducted to observe the development of cloned marbled cat embryos (marbled cat donor cells-domestic cat oocytes; MC-DC), derived from oocytes matured for 24, 36 and 42 h. The result showed that the developmental rates of MC-DC cloned embryos at the 4-8 cell and the morula stages derived from oocytes cultured for 24 h were significantly greater than those cultured for 36 and 42 h (p < 0.05). Experiment 2 was conducted to compare the fusion rate of MC-DC couplets, fused by inducing different fusion voltages, 2.1 or 2.4 kV/cm. The result showed that there was no difference in fusion efficiency between the 2.1 and 2.4 kV/cm fusion protocols. Experiment 3 was conducted to compare the developmental rate of MC-DC and domestic cat (DC-DC) cloned embryos. In vitro fertilized cat embryos served as a control. The development of MC-DC and DC-DC cloned embryos to the 4- to 8-cell, morula and blastocyst stages was not significantly different. However, the development rates at morula and blastocyst stages of control were significantly greater than those of cloned embryos (p < 0.05). Experiment 4 rabbit (RB) oocytes were used as a recipient cytoplasm for marbled cat and domestic cat cloned embryos (MC- RB and DC-RB). RB-RB cloned embryos served as a control. There were no differences in the developmental rates between MC-RB, DC-RB and RB-RB embryos. In conclusion, marbled cat fibroblast cells can be reprogrammed in domestic cat and rabbit oocytes, and by using iSCNT it might be possible to produce marbled cat offspring in the future.

Semen collection and AI in the cat are still not routine procedures. The correlation between semen quality and fertility under natural conditions is a relatively unknown field in the cat. In the present study, functional in vitro tests, such as the ability to bind and penetrate the zona pellucida or to fertilize in vitro, were used to determine fertilizing ability of sperm cryopreserved with a practical and efficient freezing protocol previously developed in our laboratory. Semen was collected by electroejaculation, evaluated for motility and diluted with Tris-glucose-citrate egg-yolk extender supplemented with Equex STM paste (0.5% v/v). After equilibration and loading into 0.25 ml straws, semen was frozen at 3.85 degrees C/min. Frozen-thawed semen was co-cultured with in vitro matured cat oocytes. Penetration rate was recorded 30 h after in vitro fertilization and cleaved zygotes were cultured in vitro until day 7. A correlation was found between sperm motility index (SMI) after thawing and semen fertilizing ability (p<0.05). In conclusion, it was demonstrated that the post-thaw motility quality, expressed as SMI, of spermatozoa frozen using the protocol mentioned above can be considered an index of the sperm ability to penetrate in vitro matured oocytes.

The ability to cryopreserve in vitro-produced feline embryos was investigated. To improve the survival rate of cryopreserved embryos, first the developmental ability of in vitro fertilized feline zygotes (after removal of intracellular lipids) was determined, followed by the post-thaw survival of cryopreserved blastocysts derived from delipidated zygotes. More than 67% of the delipidated zygotes cleaved and 36% of them developed to the morula stage. The developmental ability of delipidated zygotes to the blastocyst stage (26%) was similar to that of sham-operated (30.5%) or control embryos (31.3%). Although the survival rate of delipidated blastocysts (81.8%) after freezing and thawing tended to be higher than that of control embryos without delipidation (60.6%), rates were not significantly different between the both groups. In conclusion, in vitro-produced feline blastocysts were successfully frozen, removal of the cytoplasmic lipid content in feline zygotes did not impair their in vitro developmental competence (up to the blastocyst stage), and reduction of cytoplasmic lipids by aspiration had no apparent effects on the survival of in vitro-derived blastocysts after cryopreservation.

This study was conducted to examine whether cat spermatozoa stored in ethanol for 1 month was capable of developing into pronuclei and of supporting normal embryonic development. In vitro matured oocytes were injected with frozen-thawed spermatozoa and ethanol-stored spermatozoa. The status of oocytes and sperm nuclei was examined at 4 and 18 h after injection of spermatozoa, and the presumptive zygotes were cultured for 7 days to assess the development of oocytes injected with each storage sperm. The percentage of enlarged sperm head at 4 h after injection was higher in ethanol-stored spermatozoa than in frozen-thawed spermatozoa, but there was no significant difference in the development of oocytes and sperm nuclei at 18 h after injection between the two groups. The development of oocytes to the blastocyst stage after injection of spermatozoa was observed only in oocytes with frozen-thawed spermatozoa. Of oocytes injected with ethanol-stored spermatozoa, two (2.8%) oocytes developed to the 16-cell stage. These results indicate that cat spermatozoa stored in ethanol can decondense and form male pronuclei after intracytoplasmic injection. However, the oocytes injected with ethanol-stored spermatozoa did not have the ability to develop to the blastocyst stage.

The study involved 34 fetuses of Persian cats from eight uteruses, gestational day 58. The intraorbital muscles were analysed morphologically in respect of the location of the recti muscles, oblique muscles and retractor bulbi muscle, their length and breadth, the distance of the muscle tendon insertions from the corneal limbus and the line of insertions.

We ultrasonographically evaluated the prenatal development in cats, from Day 30 to the end of pregnancy, subjecting a group of pregnant cats (n = 8) to daily ultrasonographic examinations. The ultrasonographic images allowed us to measure the diameter of the fetal abdomen, the biparietal diameter of the fetal skull and the diameter of the fetal stomach. A correlation between these measurements and gestational age was found with a linear, parabolic and exponential regression analysis. From Days 38 to 43 after breeding we also performed fetal gender determination by evaluating the external genitalia. All queens successfully carried their pregnancies to term. This study compiled useful new data in order to clinically monitor the normal course of pregnancy in cats and to determine gestational age.

In this report, two cases of brachymelia in related neonatal cats are described. The malformations are described in view of normal limb development, which is illustrated by scanning electron microscopic images. Hypoplasia and aplasia of the distal limb segments were the main disorders in both kittens. Additional malformations in the second case included fusion of the radius and ulna, congenital patella luxation and a shortened tail. Brachymelia and similar cases of feline dysmelia are poorly documented in the existing literature. Possible causes and terminology of these developmental deformations are discussed.

Objective: To determine the relationship between parturition date and fetal skeletal mineralization detected radiographically in cats., Design: Prospective clinical trial., Animals: 31 queens and their 49 pregnancies., Procedure: Seventeen pregnant queens were radiographed with a computed radiography system every 2 to 3 days from 1 week after pregnancy was identified by abdominal palpation until parturition. Radiographs were evaluated to determine the first identifiable mineralization of 16 bony structures and teeth during each pregnancy. This information was used to establish a table of expected parturition dates on the basis of fetal mineralization. Single radiographs from an additional 32 pregnant cats were evaluated, and predictions of parturition dates were made on the basis of the mineralization table., Results: Mineralization was first detected 25 to 29 days prior to parturition (dpp). Mineralization was determined for the spinal column (22 to 27 dpp), skull (21 to 27 dpp), ribs (20 to 25 dpp), scapula (17 to 24 dpp), humerus (20 to 24 dpp), femur (19 to 23 dpp), radius (15 to 22 dpp), tibia (15 to 21 dpp), ulna (5 to 21 dpp), pelvis (8 to 20 dpp), fibula (0 to 17 dpp), tail (8 to 16 dpp), metacarpals and metatarsals (3 to 14 dpp), phalanges (0 to 11 dpp), calcaneus (0 to 10 dpp), and teeth (1 to 6 dpp). Date of parturition was predictable within 3 days in 75% of cats., Conclusions and Clinical Relevance: Identification of bony structures in the fetus is useful in estimating the time to parturition in queens.

The ability of embryos to successfully survive cryopreservation is dependent on both morphological and developmental characteristics. Domestic cat oocytes matured in vitro exhibit alterations in nuclear and cytoplasmic maturation that may affect developmental competence, particularly after cryopreservation. In Experiment 1, we evaluated the developmental competence of in vitro produced (IVM/IVF) cat embryos after cryopreservation on Days 2, 4 or 5 of IVC. In Experiment 2, in vivo viability was examined by transfer of cryopreserved embryos into recipient queens. Oocytes recovered from minced ovaries were cultured in TCM-199 with hCG/eCG and EGF at 38 degrees C in 5% O(2), 5% CO(2), 90% N(2) for 24h. In Experiment 1, after IVM/IVF, on Day 2 (n=56), Day 4 (n=48) and Day 5 (n=42) of IVC, embryos were equilibrated for 10 min at 22 degrees C in HEPES (15m M) Tyrode's (HeTy) with 1.4M propylene glycol (PG), 0.125 M sucrose (S), 10% dextran and 10% FBS, loaded into 0.25 ml straws, cooled at 2.0 degrees C/min to -6.0 degrees C and held for 10 min. After seeding, cooling resumed at 0.3 degrees C/min to -30 degrees C and after a 10 min hold, straws were plunged into liquid nitrogen (LN(2)). Straws were thawed in air for 2 min and cryoprotectant was removed by a five-step rinse consisting of 3 min each in HeTY with 0.95 M PG/0.25 M S; 0.95 M PG/0.125 M S; 0.45 M PG/0.125 M S; 0 PG/0.125 M S; 0 PG/0.0625 M S. Contemporary IVM/IVF embryos were used as nonfrozen controls (Day 2, n=14; Day 4, n=26; Day 5, n=35). After 8 days of IVC, the number of embryos developing to blastocysts was recorded and blastocyst cell numbers were counted after staining with Hoechst 33342. In Experiment 1, developmental stage did not affect the survival rate after thawing (Day 2=79%, Day 4=90%, Day 5=98%) and was not different from that of controls (Day 2=89%, Day 4=88%, Day 5=96%). Blastocyst development was similar among days both after cryopreservation (Day 2=59%, Day 4=54%, Day 5=63%) and in controls (Day 2=55%, Day 4=54%, Day 5=58%). Mean (+/-S.D.) cell number of blastocysts was slightly lower (NS) in cryopreserved embryos (Day 2=152+/-19, Day 4=124+/-20, Day 5=121+/-24) than in controls (Day 2=141+/-25, Day 4=169+/-21, Day 5=172+/-19). In Experiment 2, embryos frozen on Day 2 (n=68), Day 4 (n=49) or Day 5 (n=73) were thawed and cultured for 3, 1, or 0 days before transfer by laparotomy to 5 (mean=12.6+/-2.4), 4 (mean=12.2+/-3.7) and 6 (mean=12.0+/-1.6) recipients, respectively. Four recipients were pregnant on Day 21; two from embryos frozen on Day 4 and two from Day 5. Two live kittens were born at 66 days, a third kitten died during parturition at 64 days and a fourth pregnancy aborted by Day 45. In summary, we have shown that a controlled rate cryopreservation technique can be successfully applied to cat embryos produced by IVM/IVF. In vitro development to the blastocyst stage was not affected by the age of embryos at cryopreservation. The births of live kittens after ET of cryopreserved embryos is additional validation of progress toward applying assisted reproductive technology to preservation of endangered felids.

This study was conducted to determine the adequate medium for a serum-free culture system of domestic cat embryos produced by in vitro maturation (IVM) and fertilization (IVF). Cumulusoocyte complexes recovered from cat ovaries were matured in vitro for 24 h, and then inseminated in vitro for 12 h. After insemination, the oocytes were cultured in five media [Ham's F10, Waymouth 752/1 (Waymouth), TCM199, modified Earle's balanced salt solution (MK-1) and CR1aa], each of which contained 0.4% bovine serum albumin. There were no significant differences among the rates of fertilization of oocytes cultured in five media following IVF. The rate of oocytes/embryos developed to at least the morula stage was significantly lower (p < 0.05) in Waymouth than in MK-1, TCM199 and CR1aa. Moreover, none of the embryos cultured in Ham's F10 and Waymouth developed to the blastocyst stage. There were no differences among the rates of development to the blastocyst stage of oocytes/embryos cultured in MK-1, TCM199 and CR1aa. These results indicate that the type of serum-free medium has a major impact on in vitro development of domestic cat embryos derived from IVM/IVF, and MK-1, TCM199 and CR1aa media are suitable for in vitro culture of cat embryos in a serum-free culture system.

This work was undertaken in order to study the developmental competence of nuclear transfer feline embryos with regard to the recipient-cytoplast's age and type of somatic cells used as donor nuclei. Oocytes were recovered by mincing the ovaries in HEPES-buffered TCM-199. Selected cumulus-oocyte complexes (COCs) with compact cumulus cell mass and a dark, homogenous ooplasm were cultured for maturation in the modified medium TC-199 for 24, 35, and 43 h, and after enucleation were used as a source of recipient cytoplasts for exogenous somatic nuclei. Two experiments were carried out. In Experiment 1, the source of recipient cytoplasts was oocytes matured in vitro for 24 h (Group 1), 35 h (Group 2), and 43 h (Group 3), while the source of donor nuclei was cycling fetal fibroblasts. Somatic cell-cytoplast complexes (SC-CCs) were fused electrically by double DC pulses of 2.0 kV/cm for 15 micros. The reconstructed embryos were cultured in B2 medium for 72 h after NT, then co-cultured with BRL cells in the same medium supplemented with 10% FBS at 38.5 degrees C under 5% CO2 in air. In Groups 1, 2, and 3, the fusion rates were 71.4 (25/35), 74.6 (47/63), and 57.5% (46/80), respectively. The cleavage rates in Groups 1, 2, and 3 were 80.0 (20/25), 55.3 (26/47), and 60.8% (28/46), respectively. The development to morula and blastocyst stages was higher in Groups 1 and 2 compared to Group 3 (morula stage 14/25 (56.0%), 16/47 (34.0%), and 13/46 (28.2%); blastocyst stage 2/20 (8.0%), 4/47, (8.5%), and 0/46, respectively). In Experiment 2, the oocytes matured for 24-35 h were used as a source of recipient cytoplasts and cycling fetal fibroblasts and cumulus cells derived from mature COCs were used as a source of donor nuclei. The fusion rates were 115/193 (59.6%) versus 65/143 (45.4%) for fetal fibroblasts and cumulus cells, respectively. The cleavage rate was 72/115 (62.6%) versus 48/65 (73.8%), and the development to blastocyst stage 6/115 (5.2%) versus 5/65 (7.7%), for fetal fibroblast and cumulus cells, respectively. In conclusion, a prolonged maturation period of cat oocytes decreases developmental competence of reconstructed embryos, especially the ability to reach the blastocyst stage. The in vitro development of reconstructed embryos with either nuclei of fetal fibroblasts or cumulus cells was at approximately the same level.

Prenatal feline fetal growth and utero-placental development were ultrasonographically evaluated using an ultrasound scanner with a 10 MHz sector probe. Uterus, placenta, embryo, fetus and fetal membranes in 16 pregnant cats were monitored during the course of pregnancy; 13 subjects underwent an ovariectomy on specific days while three subjects went to term. Various anatomic structures, fixed in Carson-buffered formalin, were sectioned and then compared to ultrasound images. By ultrasound examination it is possible to evaluate every stage of the fetal development; the gestational chamber can be seen on the 10th and the embryo inside the chamber on the 14th day. By the 20th day it is possible to evaluate all the fetal membranes, and later it is possible to appreciate organs and structures such as the stomach, intestine, eyes (crystalline lens), kidneys and the cerebral choroid plexi, on the 30th, 40th, 50th, 39th and 40th day respectively. Based on our observations, it will be simpler to locate anomalies of development or pathologies during ultrasound examination of pregnant queens., (Copyright 2002 ESFM and AAFP. Published by Elsevier Science Ltd. All rights reserved.)

This study was conducted to examine the effect of the donor cat's reproductive cycle stage on in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development of oocytes recovered from ovaries that were collected and stored at 35 degrees C for a short period (1-6 h). Based on the presence or absence of follicles and corpora lutea, the ovarian pairs collected were classified into inactive, follicular, or luteal stages. Nuclear status of 161 cumulus-oocyte complexes (COCs) were examined immediately after recovery; 91.3% of the oocytes were found to be at the immature germinal vesicle (GV) stage, and 3.7% of the oocytes were at metaphase II (MII) stage. The percentage of the oocytes at the GV stage was significantly lower in the follicular stage than in the inactive stage (P < 0.01). Of the oocytes from the follicular stage, 9.1% were at MII stage. After culture for 24 h, however, the proportions of oocytes that reached metaphase I and MII were not different among the reproductive cycle stages of the ovaries collected (P > 0.05). After co-incubation with sperm, 63.1% of oocytes were fertilized, but there were no significant differences among the reproductive cycle stages of the ovaries with respect to the proportions of normal and polyspermic fertilization. However, the number of oocytes reaching cleavage stage and development to the morula and blastocyst stages from follicular stage ovaries were significantly lower (P < 0.05) than those obtained from inactive and luteal stage ovaries. These results indicate that the reproductive cycle stage of donor cat ovaries, stored at 35 degrees C, has no apparent effects on the frequencies of maturation and fertilization of oocytes, but influences developmental competence of the oocytes following IVM or IVF.

We ultrasonographically evaluated the prenatal development in cats, from the early phases to Day 30 of pregnancy, subjecting a group of pregnant cats (n = 12) to a daily ultrasonographic exam. The ultrasonographic images allowed us to measure the minor diameter of the gestational sac and the crown-rump length of the embryo/fetus. Ten subjects underwent ovariohysterectomy at specific intervals during the pregnancy, with the aim of comparing the ultrasonographic data with real data; only two subjects brought their pregnancy to term. The earliest ultrasonographic observation of the gestational sac was on Day 10 after mating, while the embryo could be measured only beginning with Day 18. This study allowed to gather useful new data in order to clinically monitor the normal course of pregnancy in cats and to date the gestational age.

We have studied the perinatal development of large myelinated axons (adult D > 10 microm) in cat ventral and dorsal lumbosacral spinal roots using autoradiography and electron microscopy (serial section analysis). These axons acquire their first myelin sheaths 2-3 weeks before birth and show nearly mature functional properties first at a diameter of 4-5 microm, i.e. 3-4 weeks after birth. The most conspicuous event during this development takes place around birth, when a transient primary myelin sheath degeneration strikes already well myelinated although short 'aberrant' Schwann cells. The aberrant Schwann cells become completely demyelinated, then measuring about 10 microm in length, and are subsequently eliminated from their parent axons. Morphometry indicates that on average 50% of the Schwann cells originally present along a prospective large spinal root axon suffer elimination. Here it should be noted that in cat lumbo-sacral spinal roots, the longitudinal growth of myelinated Schwann cells that belong to the group containing what will be the largest fibers is on average twice that of their parent axons. The elimination phenomenon is particularly striking in the dorsal roots close to the spinal cord where CNS tissue invades the root for several hundred micrometres. Our observations suggest that, once demyelinated and then eliminated, Schwann cells (i.e. aberrant Schwann cells) colonize neighbouring axons, future myelinated as well as future unmyelinated ones. In the former case the immigrant Schwann cells appear to start myelin production, possibly risking a second demyelination and elimination. We take our observations to indicate that Schwann cells in the cat, during normal development, may switch iteratively between a 'myelin-producing' and a 'non-myelin-producing' phenotype. From a functional point of view the transient presence along a myelinated axon of intercalated unmyelinated segments approximately 10 microm long, due to aberrant Schwann cells, would mean a slowing down of the action potential. The rapid disappearance of aberrant Schwann cells during the two first postnatal weeks could then explain the progressing normalization of the leg-length conduction time.

Twenty-two stages of the prenatal development of the domestic cat are described for intraspecies comparison in embryological studies. These are assigned to the 15 embryonal periods based on the Nomina Embryologica Veterinaria to make the interspecies comparison possible.

The effects of protein supplementation in culture medium on development to the hatching and hatched blastocyst stages of cat in vitro-fertilized embryos were investigated. In the first experiment, presumptive zygotes derived from in vitro maturation and in vitro fertilization (IVF) were cultured in modified Earle's balanced salt solution (MK-1) supplemented with 0.4% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS) for 9 days. There were no significant differences between the BSA and FBS groups with respect to the proportion of cleavage and development to the morula and blastocyst stages of zygotes. However, the presence of FBS in the medium enhanced development to the hatching blastocyst stage of zygotes compared with the BSA group (31.4% v. 7.8%). Moreover, 2.9% of zygotes cultured with FBS developed to the hatched blastocyst stage. The mean cell number of blastocysts derived from zygotes cultured with FBS was significantly higher (P<0.01) than that from zygotes cultured with BSA (136.6 v.101.5). In the second experiment, embryos at the morula orblastocyst stage, which were produced by culturing in MK-1 supplemented with 0.4% BSA after IVF, were subsequently cultured in MK-1 with 0.4% BSA or 5% FBS. Significantly more morulae developed to the blastocyst (P<0.05) and hatching blastocyst stages (P<0.01) in the FBS group than in the BSA group (71.5% and 53.6% v. 44.9% and 6.0%, respectively). Although none of the morulae cultured with BSA developed to the hatched blastocyst stage, 11.5% of morulae cultured with FBS developed to the hatched blastocyst stage. Moreover, the proportion of development to the hatching blastocyst stage of blastocysts was significantly higher (P<0.01) in the FBS group than in the BSA group (68.7% v. 9.8%). None of the blastocysts cultured with BSA developed to the hatched blastocyst stage, whereas 7.3% of blastocysts cultured with FBS developed to the hatched blastocyst stage. The results of the present study indicate that supplementation with FBS at different stages of early embryo development promotes development to the hatching and hatched blastocyst stages of cat IVF embryos.

The pre- and post-natal development of the feline claw was studied in 22 feline fetuses with a crown-rump length (CRL) ranging from 40 to 160 mm, in six kittens up to an age of 22 days, and in four adult cats. In fetuses up to a CRL of 75 mm, the characteristic shape of the feline claw was developed. Segment-specific dermal modifications in the various segments, especially the dorsal ridge, started to develop in fetuses with a CRL between 75 and 105 mm. Modifications of the papillary body in the different claw segments took place in the last third of prenatal development and were continued postnatally. At first, the basal lamina became wavy, followed by the formation of small dermal microridges, which would be enlarged to dermal ridges and lamellae. In the claw of adult cats, the papillary body was very small. The dermal tissue of the proximal part of the coronet formed low microridges with short papillae originating on and between these low ridges. In the wall segment, dermal microridges were formed which were arranged in a parallel fashion, and in the distal part of the wall, short dermal micropapillae arose on the crest of each microridge. In the sole segment, thin dermal lamellae were developed. The sequence of papillary body development and the varying conformations of the papillary body in the different segments of the feline claw are compared to those in the nail, the canine claw and hooves.

The purpose of this study was to evaluate the ability of cat oocytes, at different stages of maturation, to survive after cryopreservation and to assess their subsequent development following IVM and IVF. In the initial toxicity trial, immature oocytes were exposed to different concentrations of DMSO and ethylene glycol (EG). Resumption of meiosis and metaphase II were evaluated after removal of the cryoprotectant and IVM. The highest rates of resumption of meiosis (51.4%) were achieved after exposure to 1.5 mol l(-1) of cryoprotectants, and no difference was observed with control oocytes. Metaphase II was obtained in 25.7% (P<0.01) and 22.9% (P<0.005) of oocytes exposed to 1.5 mol l(-1) of DMSO and ethylene glycol, although at lower rates than in control oocytes (54.4%). On the basis of this finding, 1.5 mol l(-1) of cryoprotectant was chosen for freezing cat oocytes at the germinal vesicle stage (immature) or at metaphase II stage (mature). Post-thaw viability was assessed by the evaluation of the embryo development in vitro. After fertilization, mature oocytes frozen in ethylene glycol cleaved in better proportions (38.7%) than immature oocytes (6.8%, P<0.001), and no differences were observed in the cleavage rate of oocytes frozen at different maturation stages with DMSO (immature 12.8%; mature 14.1%). Embryonic development beyond the 8-cell stage was obtained only when mature oocytes were frozen with ethylene glycol (11.3%). This study suggests that cryopreserved cat oocytes can be fertilized successfully and that their development in vitro is enhanced when mature oocytes are frozen with ethylene glycol. The stage of maturation may be a key element in improving cat oocyte cryopreservation.

The bcl-2 family of genes encodes proteins that influence apoptosis. In the present immunohistochemical study, the topographic distribution of bcl-2 protein was examined in healthy feline fetal, neonatal, and adult tissues, a feline renal cell line, and feline tumors obtained from a veterinary hospital. The topographic distribution of bcl-2 in healthy tissues was similar to that described in human tissues. In lymphoid tissues, follicular mantle cells strongly expressed bcl-2. In complex and differentiating epithelium, bcl-2 expression was detected in stem cell and proliferation zones. Bcl-2 expression was also detected in lower crypts of the intestine and in skin basal layers. The feline Crandell kidney cells expressed bcl-2 diffusely throughout the cytoplasm. Of 180 tumors examined, bcl-2 was expressed almost uniformly in cutaneous basal cell tumors, thyroid adenomas, and mammary carcinomas and in 50% of the lymphomas examined. Bcl-2 may play a role in blocking apoptotic cell death in a broad range of normal feline tissues, whereas dysregulated bcl-2 may extend the life of certain tumors or render certain tumors resistant to therapy because most chemotherapeutic and radiotherapeutic agents eliminate tumor cells by triggering apoptosis.

Development of cat oocytes following intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) was compared in two experiments. Domestic cat donors (used as a model for wild felids) were treated with 150 IU equine chorionic gonadotrophin (eCG) on treatment day 1 or a total of 10-15 IU of follicle-stimulating hormone (FSH) over four days, followed by 100 IU human chorionic gonadotrophin (hCG) on day 5 and follicular aspiration 24-26 h later. A jaguarundi (Herpailurus yaguarondi) female was stimulated twice with FSH (20 IU) or eCG (300 IU) and hCG (250 or 300 IU) before oocyte recovery. After storage at 4 degrees C, domestic cat semen was washed and processed. For ICSI, denuded oocytes were each injected with an immobilised spermatozoon. IVF oocytes were co-incubated with 5 x 10(4) motile spermatozoa/0.5 ml for 4-6 h. Noncleaving oocytes were fixed and stained 24-28 h after injection or insemination. Presumptive zygotes were cultured before transfer on day 5 (experiment I only) or evaluation on day 7 (experiments I and II). In experiment I, fertilization frequency was 67.9% (72/106) and 58.1% (122/210) for IVF and ICSI oocytes, respectively (P > 0.05). Most noncleaving ICSI oocytes (71/88, 80.7%) at 24 h were at metaphase II, of which half (35/71, 49.3%) had an activated spermatozoon (n=4) or premature chromatin condensation (PCC, n=31) of the sperm head. All 69 day 7 IVF embryos developed to morulae (> 16-cells, 46.7%) or blastocysts (53.3%), and 59/63 (93.7%) ICSI embryos reached the morula (50.8%) or blastocyst (42.9%, P > 0.05) stage. Mean cell number in IVF and ICSI embryos was 136 and 116 (P > 0.05); morulae had 77 and 46 (P < 0.05) and blastocysts had 187 and 209 (P > 0.05) cells, respectively. After transfer of 10 or 11 day 5 ICSI morulae to each of four recipients, a total of three kittens were born to two dams at 66 or 67 days. Of 18 fair-to-good quality oocytes recovered from a jaguarundi on two occasions, 10 (55.6%) embryos were produced by ICSI with fresh (n=5) or frozen (n=5) conspecific spermatozoa, but no jaguarundi kittens were born after transfer of these embryos to domestic cat recipients. In experiment II, cleavage frequency following IVF (15/17, 88.2%) and ICSI (31/38, 81.6%) was higher (P < 0.05) than following sham ICSI (13/35, 37.1%). Mean cell number (27 cells) and blastocyst development (0%) on day 7 was lower (P < 0.05) in the sham ICSI group than in the ICSI group (45 cells, 15.6% blastocysts) which, in turn, was lower (P < 0.05) than the IVF group (94 cells, 46.7% blastocysts). We have demonstrated that ICSI can be applied successfully in domestic felids and suggest that the technique will effectively augment other biotechniques being developed for enhancing reproduction in endangered felids.

The correlated activity of developing retinal ganglion cells is essential for the reorganization and refinement of retinogeniculate projections. Previous studies have uncovered marked changes in the spiking properties of retinal ganglion cells during this period of reorganization; however, a full understanding of the changes in the underlying ionic conductances has yet to be obtained. To this end, the whole-cell configuration of the patch-clamp technique was used to record currents conducted by voltage-gated calcium channels in 83 dissociated cat retinal ganglion cells obtained from animals aged between embryonic day 34 and postnatal day 105. Calcium currents, magnified by using barium as the major charge carrier, were isolated by substituting choline for Na+ in the bathing solution and Cs+ for K+ in the electrode solution. Three voltage-gated Ca2+ conductances were identified based on their voltage dependence and kinetics of activation and inactivation: a transient low-voltage-activated conductance, a transient high-voltage-activated conductance and a sustained high-voltage-activated conductance. During the developmental period examined there were significant increases in the densities of all three conductances, as well as significant changes in some of their activation and inactivation properties. These findings, together with those reported previously for the voltage-gated Na+ and K+ conductances, are related to the generation of excitability in developing retinal ganglion cells during a period critical to the normal development of the visual system. Furthermore, while the sustained high-voltage-activated conductance was present in all of the retinal ganglion cells observed, only about 72% expressed the transient high-voltage-activated current. During the developmental period examined, there was also an increase in the proportion of cells expressing the transient low-voltage-activated conductance. This, along with our previous finding that retinal ganglion cells heterogeneously express different types of voltage-gated K+ channels, strongly suggests that the spiking patterns observed in different classes of retinal ganglion cell may be due, in part, to their intrinsic membrane properties.

The aim of the present study was to determine, histochemically, the onset and location of production of preduodenal lipase in fetal, suckling, weaned and adult cats. Strong enzymatic activity was localized in the surface mucous cells of the gastric mucosa in animals at postpartal day 1 after ingestion of milk. Activity of gastric lipase persisted as long as animals were nursed. No gastric lipase could be demonstrated in weaned and adult cats. Lingual lipase was not found at any developmental stage examined. Thus, in the newborn cat, lipase of the gastric mucosa is responsible for milk fat lipolysis.

Feline herpesvirus type 1 (FHV-1) gD-expressing Sf9 cells adhered to two feline cell lines, but not to the porcine, bovine, or canine cell lines tested. In addition, this adhesion activity was inhibited by a monoclonal antibody against FHV-1 gD. These results showed that the FHV-1 gD might bind to a specific-molecule(s) on the surface of feline cell lines. We discussed a possible importance of the FHV-1 gD in host cell restriction to FHV-1 infection.

There are five stages in the development of the cat's gastric glands: 1. During the stage of the indifferent epithelium from day 19 to day 24, the anlage of the stomach develops with all layers; 2. The stage of gland formation from day 24 to day 41 is the beginning of the gland buds. They develop in connection with endocrine cells on day 34 into primitive oxyntic and primitive mucous cells. The latter form the basis for all other cells, including the surface mucous cells; 3. During the stage of gland evagination from day 42 to 55, the anlagen are separated into primitive pits and tubules, while the cells continue to differentiate and the first intermediate cells are seen; 4. The stage of gland branching from day 56 to birth is characterized by the formation of additional glands at the bottom of the pits which change the ordinary anlagen into branched glands. During this stage, the cardiac glands are formed; 5. In the stage of gland maturation from birth to the 9th week, the peptic cells are formed and the glands start functioning. The oxyntic cells show carbonic-anhydrase activity and signs of acid secretion, and, between the weeks 4 and 8, the peptic cells contain pepsinogen, producing a negative reaction to PAS and a positive reaction to HID. Mucous cells and mucous neck cells produce PAS- and AB-positive mucin.

A striking feature of the internal capsule during early development is that it is full of small neurones. Later, this group of neurones, called the perireticular thalamic nucleus, appears to have reduced in size, and only a few scattered cells are seen. In an effort to understand better the developmental history of the perireticular nucleus this study examines: i) the period of cell generation in the nucleus, ii) the magnitude of cell loss in the nucleus, and iii) the subsequent fate of cells in the nucleus during development. The perireticular cells are generated very early in development, being among the first generated in the thalamus (rats: E13-14; cats: E21-30). In rats, the first perireticular cells are generated at about the same developmental stage as the first subplate cells, which are among the first generated cells of the cortex: in cats, the first perireticular cells are generated well before those in the subplate (E24-30). In rats, the number of perireticular cells during developmental peaks at P5 (approximately 30,000) and then declines sharply (approximately 98%) by P15 (approximately 750), when adult-like patterns are seen. This dramatic loss of perireticular cells is due to both cell death and a migration of cells into the adjacent globus pallidus. The majority of the perireticular cells which migrate into the globus pallidus, however, are likely to die also. The presence of pyknotic profiles (indicators of dying cells) in the rat perireticular nucleus points to cell death as a contributor to the reduction in cell number during development. In this study, a period of relatively high pyknotic profile incidence (number of pyknotic cells per 1,000 "living" cells) is recorded in the perireticular nucleus over a 5 day period, from P2 to P7 (13.5-15.5). Similar values and patterns are recorded in the reticular nucleus and globus pallidus, except that in these structures, a period of relatively high pyknotic profile incidence (15-20) occurs over a shorter period (3 days; P2-5). Previous studies have suggested that some perireticular cells migrate into and settle within the adjacent globus pallidus. This study, with the use of long-term survivals after tracer injections in rats, shows that none (or very few) of these perireticular cells which migrate into the globus pallidus survive into more mature postnatal stages. Tracer (biotinylated dextran) was injected into the sensory nuclei of the dorsal thalamus at early stages (P7) and the rats were allowed to survive for either a day thereafter (to P8) or until well after the period of cell death was complete (to P16 or P21). In the short-term survivals (to P8), there are many dextran-labelled cells seen in the globus pallidus and in the perireticular nucleus. In the long-term survivals (to P16 or P21), by contrast, there are no dextran-labelled cells apparent in the globus pallidus or in the perireticular nucleus. It is likely that these cells in the globus pallidus, as with those in the perireticular nucleus, undergo cell death during development.

Ordered neuronal connections in mature brains are thought to be sculpted from initially diffuse projections by elimination of inappropriate projections and strengthening of appropriate ones. Although evidence suggests that neuronal activity plays a role in these processes, the mechanism behind the modification of neuronal connections remains obscure. To gain insight into the mechanisms of axonal elimination and projection strengthening, we examined the morphology of individual axons that were to be eliminated as well as the consequences of partial denervation. While corticorubral projections in adult cats are thought to be uncrossed, early in postnatal development and after early unilateral lesions to the sensorimotor cortex, however, a significant amount of crossed corticorubral projections occurs. We examined the morphology of individual corticorubral axons in fetal cats and kittens from embryonic day 59 to postnatal day 48 and those that had received early unilateral lesions to the cortex, by serial reconstruction of Phaseolus-vulgaris-leucoagglutinin- or biocytin-labeled axons. For about 2 weeks during pre- and postnatal development, crossed axons remained simple in morphology, with few branches. Thereafter, they showed an increase in branch number, but then began to show fewer branches again. Axons and their collaterals were found in nonrestricted areas of the red nucleus (RN) throughout the period of observation, indicating that axons can sit at an inappropriate target for weeks but fail to ramify. In contrast, crossed corticorubral axons in kittens with cortical lesions showed terminal-arbor-like structures in the RN region that are in mirror symmetry to topographically appropriate areas in the ipsilateral RN, although some showed simple morphology without arbors. These complicated forms of morphology of individual axons during development and after partial denervation may not be explained by a simple activity-dependent mechanism.

Much is known about the morphological development of mammalian retinal ganglion cells. However, relatively little is understood about the mechanisms that direct dendritic development and determine ganglion cell morphology, mosaic organization, and foveal development. In the following, we review current data on primate retinal ganglion cell development and integrate it with information from other species into a general model of dendritic development. Furthermore, we propose that this model not only explains ganglion cell dendritic development, but also accounts for the establishment of retinal ganglion cell mosaics and the timing of foveal formation.

Serum hormones, corpus luteum (CL) progesterone, and CL LH receptors were characterized and interrelated to ovarian follicle and CL number and preimplantation embryo quality/survival in the cat. Blood samples were collected from queens ovariohysterectomized at 64 (n = 7), 76 (n = 11), 100 (n = 8), 124 (n = 7), 148 (n = 6), or 480 (n = 8) h after first copulation (3-times-daily matings on Days 2 and 3 of estrus). Ovarian CL were enucleated, weighed, and bisected; one hemi-CL was assayed for progesterone and the other for LH receptors. Serum was assessed for estradiol-17 beta and progesterone. Serum estradiol-17 beta concentrations did not return (p > 0.05) to baseline approximately 20 pg/ml) until 124 h after first copulation, whereas serum progesterone began to increase (> 1 ng/ml) by 76 h, was elevated (p < 0.05) by 124 h, and continued to rise thereafter. Serum progesterone was highly correlated with CL mass and LH receptor and progesterone concentration (range, r = 0.69-0.82; p < 0.01). CL progesterone and LH receptor concentrations were similar (p > 0.05) at 64 and 76 h, and both increased (p < 0.05) at subsequent time intervals and were correlated closely (r = 0.65; p < 0.01). Although ovarian CL were distinct and well organized by 64 h, pronounced elevations in serum progesterone and CL LH receptors and progesterone did not occur until at least 36 h later. The rapid increase in serum progesterone concentrations between 100 and 148 h was related to accelerated LH receptor synthesis and CL progesterone production and not entirely to enhanced CL growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Pregnancy rates are low and litter sizes generally small when assisted reproduction techniques are used in gonadotropin-treated felid (cat) species. A prerequisite to determining whether or not abnormal morphological changes in the uterine lumen or corpus luteum (CL) are related to this reproductive failure is the documentation of normal histological kinetics during natural embryogenesis. This study characterized the histological changes of the endometrium and ovarian CL during the early stages of preimplantation embryonic development in the naturally estrous, mated queen. The purpose was to 1) develop a system for dating the cat endometrium and CL of early pregnancy; 2) document the frequency of abnormal uterine and CL history under natural mating conditions; and 3) compare histological traits of queens producing good- vs. poor-quality embryos. Naturally estrous, mated queens were ovariohysterectomized at 64 h (n = 8), 76 h (n = 11), 100 h (n = 8), 124 h (n = 7), 148 h (n = 6), or 480 h (n = 8) after first copulation. Embryos collected from oviductal and uterine flushings were graded for quality, and uteri and ovaries were fixed in formalin. Fixed tissue sections were stained and multiple histological traits described for each uterine (endometrial height, endometrial vacuolation, percentage of glandular cells with subnuclear vacuoles, number of mitoses, nuclear-to-cytoplasmic ratio) and ovarian (presence of tertiary follicles, vacuolation of luteal cell cytoplasm, luteal cell shape) sample. Modest histological changes were observed at each time point, and these were documented in detail. The most prominent modifications occurred at 124 h after first copulation and included thickening of the endometrium, straightening of the glands, increased cytoplasmic vacuolation, and increased epithelial height. Of 15 queens failing to produce good-quality embryos, only 4 expressed unusual histological characteristics; and 3 of 25 queens producing only high-quality embryos exhibited abnormal uterine or CL cellular integrity. Therefore, aberrant histological changes are not primarily responsible for failure of the naturally estrous, mated queen to produce good-quality embryos. Furthermore, a normative database now is available to date the endometrium and CL of early pregnancy and to examine the impact of exogenous gonadotropins and assisted techniques on uterine/CL structure and function.

In the adult cat, neuropeptide Y (NPY) immunoreactivity (IR) is found within a subgroup of gamma-type ganglion cells and a large group of regularly arrayed amacrine cells. To examine the development of these two cell groups, we charted the appearance and maturation of neuropeptide Y immunoreactivity in the pre- and post-natal cat retina. Neuropeptide Y immunoreactivity is first observed at the central retina within the ganglion cell layer on embryonic day 46, and immunoreactivity within amacrine cells of the inner plexiform layer is present by E50. The number of immunoreactive profiles reaches the adult level in the amacrine population first (around P7), while the ganglion cell population shows a protracted development, with new cells being added until the third postnatal week. NPY-immunoreactive profiles in the ganglion cell layer were confirmed to be ganglion cells by retrograde labeling in both pre- and post-natal animals. Thus, neuropeptide Y-immunoreactive ganglion cells and amacrine cells attain their mature state with very different timecourses, although both cell groups initially follow a central to peripheral pattern of development. Interestingly, NPY expression within the ganglion cell population is temporally correlated with retinal synaptogenesis in the inner plexiform layer. As in the adult cat, NPY-immunoreactive ganglion cells never show a regular distribution during development, while NPY-IR amacrine cells are always distributed regularly even at the earliest ages. The prenatal presence of a regular distribution of NPY-IR amacrine cells suggests that these cells may participate in establishing the ganglion cell mosaics that appear during postnatal development.

Sensory-dependent modification of cortical morphology is one component of the cortical plasticity that occurs during the critical period for ocular dominance changes. In this study, we used dark-rearing to examine the sensory dependency of subplate neuron death and the quantity of microtubule-associate protein 2 (MAP2)-positive dendrites. Kittens reared in total darkness until the peak of the critical period had fewer laterally extended MAP2-positive dendrites than age-matched normal kittens. This reduction was found in layer IV but not in layer V. Subsequent exposure to light for 10 days after dark-rearing was sufficient to bring the number of MAP2-positive dendrites to the normal level. Contrarily, dark-rearing did not prevent subplate neurons from dying. Exposure to light after dark-rearing did not increase the number of potential dying neurons. These results show that the quantity of MAP2-positive dendrites is sensory-dependent; however, the death of the subplate neurons is not. Therefore, the death of subplate neurons is probably not directly involved in sensory-dependent modifications of synaptic connections. The possible involvement of laterally extended MAP2-dendrites in visual plasticity is discussed.

The prenatal development of the cat's mandibular and parotid gland was examined by means of serial section of 36 cat embryos at 14-62 days of development. Both glands were excised from the epithelium of the primitive oral cavity and branched up to day 36 of the branching phase into a specific connective tissue. This tissue contained besides fine collagenous fibres, a high amount of proteoglycans. In the subsequent separation phase, ducts and acini differentiated themselves in primitive lobules which were separated by connective tissue. In the prenatal differentiation phase, from about day 50 up to birth, intercalated ducts and striated ducts were formed. In the acini, mucous and serous cells contained different amounts of complex carbohydrates. This secretory component changed shortly before birth.

The prenatal development of the clavicular area was studied in two species: the domesticated sheep, which lacks a clavicle, and the cat, with a nonfunctional, rudimentary clavicle. A morphological and computerized morphometric study of the clavicle was performed in 18 cat embryos between 25 and 48 days of gestation, and in 12 sheep embryos of 37-45 days. One group of embryos was processed with double staining in toto according to Hanken and Wassersug in 1981. The other group was examined by histological techniques: hematoxylineosin-Alcian blue and picrosirius. In both species, clavicular ossification is delayed (27% of gestation time elapsed in sheep and 53% in cats) compared to 16% in humans. Histological and morphological differences in shape and length of the clavicle were observed in both species. The clavicle is transient in sheep, whereas in the cat it persists with little change. In neither species does secondary cartilage develop. In cats, the periosteum is well developed with active osteoblasts, whereas in sheep the clavicle is surrounded by a single layer of epitheloid cells and the periosteum is less developed and contains osteoclasts. These results suggest that the morphogenetic pattern for the clavicle is altered at about day 34 in cats and day 40 in sheep, and is subsequently partly inhibited in the former and blocked in the latter.

Development of in vitro-fertilized (IVF) cat embryos was compared to that of naturally produced cat embryos in vivo and in vitro. To obtain in vivo-fertilized embryos, queens were mated three times daily on the second and third days of natural estrus and ovariohysterectomized at 64, 76, 100, 124, or 148 h after the first copulation. Embryos were flushed from the reproductive tract, evaluated for developmental stage, and cultured. For IVF, oocytes from gonadotropin-stimulated queens were inseminated with electroejaculated cat sperm in Ham's F-10 and evaluated for fertilization (cleavage to > or = 2 cells) at 30 h. In vitro development of embryos fertilized in vivo (n = 109) and in vitro (n = 46) was evaluated every 24 h for up to 10 days. High-quality embryos recovered at 64, 76, 100, 124, and 148 h after the first copulation were typically 1 to 2 cells (13 of 20), 5 to 8 cells (18 of 28), 9 to 16 cells (14 of 24), morulae (15 of 21), and compact morulae (11 of 18), respectively, suggesting blastomere cleavage once per day in vivo after the first three rapid cell divisions. A similar developmental rate to the morula stage (p > or = 0.05) was achieved in vitro by embryos derived from both in vitro and in vivo fertilization. Additionally, the proportion (p > or = 0.05) of in vivo-generated embryos (2 to 16 cells) that developed to morulae (64 of 83; 77.1%) was similar to that of IVF embryos (28 of 46; 60.9%). However, none of the IVF embryos (0/46), but 70.6% (77 of 109) of the in vivo-produced embryos, achieved blastocyst formation in culture (p < or = 0.05). Furthermore, 66.2% (51 of 77) of these blastocysts exhibited zona hatching. Incidence of morula and blastocyst formation in the in vivo group was influenced by stage of the embryo at collection. Embryos that were at the 9- to 16-cell stage at recovery were more likely (p < or = 0.05) to achieve morula or blastocyst status and emerge from the zona pellucida than younger-stage counterparts. In summary, the in vivo and in vitro growth rate of cat embryos produced after natural mating was comparable to that of embryos fertilized and cultured in vitro. However, developmental ability to the blastocyst stage was superior for embryos produced in vivo after natural mating.(ABSTRACT TRUNCATED AT 400 WORDS)

In vivo embryogenesis, embryo migration, and survival were studied in the domestic cat. Queens were naturally mated (three times daily) on the second and third days of behavioral estrus and, if ovulation occurred, ovariohysterectomized at 64 (n = 8), 76 (n = 11), 100 (n = 8), 124 (n = 7), 148 (n = 6), and 480 h (n = 8) after first copulation. Of 52 cats mated, 48 (92.3%) ovulated (as evidenced by the presence of ovarian CL), and of these, 38 (79.2%) either produced good-quality embryos or had implantation sites. From the remaining cats, only unfertilized oocytes (n = 5), degenerating embryos (n = 4), or no oocytes/embryos (n = 1) were recovered. Embryos at 64, 76, 100, and 124 h after the first copulation typically were 1 to 4 cells (17 of 20; 85.0%), 5 to 8 cells (18 of 28; 64.3%), 9 to 16 cells (14 of 24; 58.3%), and morulae (15 of 21; 71.4%), respectively; all were within the oviducts. At 148 h, embryos primarily were compact morulae or early blastocysts (15 of 18; 83.3%), and all were within the uterus. For the preimplantation groups, the overall recovery of embryos plus oocytes per CL was 80.6%, and the mean (+/- SEM) numbers of CL and embryos were 64 h, 4.8 +/- 0.3, 3.1 +/- 0.8; 76 h, 4.7 +/- 0.3, 3.9 +/- 0.6; 100 h, 5.8 +/- 0.5, 3.3 +/- 0.8; 124 h, 4.4 +/- 0.5, 4.0 +/- 0.6; and 148 h, 6.5 +/- 1.1, 3.7 +/- 0.7, respectively. Cats in the 480-h group produced a mean of 5.6 +/- 0.5 CL and 3.9 +/- 0.5 implantation sites. In six of eight cats in this group, there was a disparity between CL number on a given ovary and number of implantation sites in the ipsilateral horn, supporting the concept of transuterine embryo migration. In summary, results indicated that 1) more than 90% of cats ovulated following this multiple mating regimen, but approximately 21% of these failed to produce any fertilized or viable embryos; 2) embryo developmental rate in vivo was biphasic, with a rapid cleavage rate to the 5- to 8-cell stage followed by a slower cleavage rate to the morula stage; 3) cat embryos entered the uterus as compact morulae or early blastocysts approximately 5.5 days after the first copulation; and 4) on the basis of implantation/CL ratio, approximately 30% of all ovulated cat oocytes underwent either fertilization failure or preimplantation embryonic mortality.

This paper demonstrates the structure and development of the layer of mm. contrahentes in Felis catus (Carnivora). Ontogenesis of the muscle layer was studied on 35 histological series of embryonal extremities and by microdissections of palmar musculature in the domestic cat. It was found that three mm. contrahentes were formed passing to the first, second and fifth digits. A compact anlage of the muscle layer (similar to that in embryos of many mammalian species) has been observed only in foetuses of 15-16 mm crown-rump length. Differentiation of the anlagen of the individual muscles takes place in the foetuses of 20-23 mm crown-rump length.