In vitro fertilization and sperm cryopreservation in the black-footed cat (Felis nigripes) and sand cat (Felis margarita).

Studies of in vitro fertilization (IVF) and sperm cryopreservation have been conducted in several small cat species, but virtually no data exist for black-footed cats (Felis nigripes) (BFCs) or sand cats (Felis margarita) (SCs). The objectives of this study were 1) to compare in vitro motility and acrosome status of fresh and cryopreserved (frozen in pellets on dry ice or in straws in liquid nitrogen vapor) BFC and SC spermatozoa cultured in feline-optimized culture medium (FOCM) or Ham F-10, 2) to assess ovarian responsiveness in BFCs and SCs following exogenous gonadotropin treatment and laparoscopic oocyte recovery, and 3) to evaluate the fertility of fresh and frozen-thawed spermatozoa from both species using homologous and heterologous (domestic cat oocytes) IVF in the two culture media. Motility and acrosomal integrity of fresh and frozen-thawed spermatozoa from BFCs and SCs were similar (P > 0.05) in both media during 6 h of culture. Although effects were more pronounced in SCs, cryopreservation in straws was superior (P < 0.05) to cryopreservation in pellets for both species. Gonadotropin stimulation produced approximately 16 ovarian follicles per female, and >80% of recovered oocytes were of optimal (grade 1) quality. The BFC and SC spermatozoa fertilized 60.0%-79.4% of homologous and 37.7%-42.7% of heterologous oocytes in both culture media, with increased (P < 0.05) cleavage of homologous (SC) and heterologous (BFC and SC) oocytes in FOCM. These results provide the first information to date on the gamete biology of two imperiled cat species and further our capacity to apply reproductive technologies for their conservation.

Fecal endocrine profiles and ejaculate traits in black-footed cats (Felis nigripes) and sand cats (Felis margarita).

Information regarding the reproductive biology of black-footed cats (BFC) and sand cats (SC) is extremely limited. Our objectives were to: (1) validate fecal hormone analysis (estrogens, E; progestagens, P; androgens, T) for noninvasive monitoring of gonadal activity; (2) characterize estrous cyclicity, ovulatory mechanisms, gestation, and seasonality; and (3) evaluate male reproductive activity via fecal androgen metabolites and ejaculate traits. In both species, the estrous cycle averaged 11-12 days. In BFC (n=8), estrus lasted 2.2+/-0.2 days with peak concentrations of E (2962.8+/-166.3 ng/g feces) increasing 2.7-fold above basal concentrations. In SC (n=6), peak concentrations of E (1669.9+/-83.5 ng/g feces) during estrus (2.9+/-0.2 days) were 4.0-fold higher than basal concentrations. Nonpregnant luteal phases occurred in 26.5% (26 of 98) of BFC estrous cycles, but were not observed in SC (0 of 109 cycles). In both species, P concentrations during pregnancy were elevated (32.3+/-3.0 microg/g feces BFC; 8.5+/-0.7 microg/g feces SC) approximately 10-fold above basal concentrations. Fecal T concentrations in males averaged 3.1+/-0.1 microg/g feces in BFC and 2.3+/-0.0 microg/g feces in SC. Following electroejaculation, 200 to 250 microl of semen was collected containing 29.9 (BFC) to 36.5 (SC)x10(6) spermatozoa with 40.4 (SC) to 46.8 (BFC)% normal morphology. All females exhibited estrous cycles during the study and spermatozoa were recovered from all males on every collection attempt, suggesting poor reproductive success in these species may not be due to physiological infertility.

Assisted reproduction mediated resurrection of a feline model for Chediak-Higashi syndrome caused by a large duplication in LYST.

Chediak-Higashi Syndrome (CHS) is a well-characterized, autosomal recessively inherited lysosomal disease caused by mutations in lysosomal trafficking regulator (LYST). The feline model for CHS was originally maintained for ~20 years. However, the colonies were disbanded and the CHS cat model was lost to the research community before the causative mutation was identified. To resurrect the cat model, semen was collected and cryopreserved from a lone, fertile,  CHS carrier male. Using cryopreserved semen, laparoscopic oviductal artificial insemination was performed on three queens, two queens produced 11 viable kittens. To identify the causative mutation, a fibroblast cell line, derived from an affected cat from the original colony, was whole genome sequenced. Visual inspection of the sequence data identified a candidate causal variant as a ~20 kb tandem duplication within LYST, spanning exons 30 through to 38 (NM_001290242.1:c.8347-2422_9548 + 1749dup). PCR genotyping of the produced offspring demonstrated three individuals inherited the mutant allele from the CHS carrier male. This study demonstrated the successful use of cryopreservation and assisted reproduction to maintain and resurrect biomedical models and has defined the variant causing Chediak-Higashi syndrome in the domestic cat.

The role of transcription in EGF- and FSH-mediated oocyte maturation in vitro.

Understanding mechanisms responsible for meiotic resumption in mammalian oocytes is critical for the identification of strategies to enhance developmental competence of in vitro-matured oocytes. Improvement of in vitro oocyte maturation systems is dependent on a better understanding of mechanisms that regulate oocyte maturation both in vivo and in vitro as well as on the identification of methods to manipulate the meiotic progression of oocytes matured in vitro in a physiological manner. The purpose of this review is two-fold: first, to examine the mechanisms that underlie the acquisition of oocyte developmental competence and regulation of oocyte maturation in vivo and in vitro; second, to present data examining the role of transcription in mediating the ability of EGF and FSH to induce oocyte maturation in vitro. Results presented support the conclusions that (1) EGF-induced oocyte maturation does not require nascent gene transcription in both mice and domestic cats; (2) FSH requires gene transcription to induce oocyte maturation in both species; (3) EGF must be present in the maturation medium to optimize the effectiveness of FSH to promote oocyte maturation; (4) the mechanism used by FSH to induce oocyte maturation in vitro appears to predominate over that used by EGF when both EGF and FSH are present in maturation medium used for either murine or feline cumulus oocyte complexes.

A comparative analysis of metabolism and viability in porcine oocytes during in vitro maturation.

The importance of oocyte quality cannot be overstated, because it impacts all subsequent events during development of the embryo, the fetus and even the resulting offspring. Oocyte metabolism plays a critical role in supporting developmental competence via multiple mechanisms. It is beginning to be understood that metabolic pathways not only affect cytoplasmic maturation but may control nuclear maturation as well. A complete understanding of the precise roles that metabolism plays in determining oocyte quality is crucial for developing efficient in vitro maturation systems to support acquisition of oocyte competence. To date, this pursuit has not been entirely successful. Work in our laboratory on porcine oocyte metabolism has elucidated some of the intricate control mechanisms at work within the oocyte, not only for energy production, but also encompassing progression of nuclear maturation, mitochondrial activity and distribution, and oxidative and ionic stresses. We hypothesize that by utilizing oocyte metabolic data, we can develop more appropriate in vitro maturation systems that result in increased oocyte and embryo developmental competence.

Reduced polyspermic fertilization of porcine oocytes utilizing elevated bicarbonate and reduced calcium concentrations in a single-medium system.

The development of efficient systems for in vitro production of porcine embryos has been hampered by a high incidence of polyspermic fertilization. A recently developed single-medium system for porcine in vitro maturation (IVM), IVF and in vitro embryo culture (IVC) (Purdue Porcine Medium; PPM) was modified with elevated bicarbonate (44 mM) and reduced calcium concentrations (1.7 mM) for IVF (PPMfert.2). Oocyte penetration was evaluated after maturation in PPMmat (0.5 mg mL(-1) hyaluronan, 0.6 mM cysteine, 10 ng mL(-1) epidermal growth factor (EGF), 0.1 U mL(-1) porcine LH and FSH, and 1 x Minimal Essential Medium (MEM) vitamins) and fertilization (5 h with 5 x 10(5) sperm mL(-1)) in either PPMfert.2 or mTBM (20 mM Tris, 0.0 mM bicarbonate, 7.5 mM calcium). Embryonic development (cleavage and blastocyst stages) was assessed after culture in PPM1 and PPM2. Although penetration was lower in PPMfert.2 (69.9%) compared with mTBM (83.9%), 48.8% of penetrated oocytes were fertilized normally in PPMfert.2 compared with only 27.8% normal fertilization in mTBM. More oocytes cleaved in PPMfert.2 (77.9% v. 53.7%), but development to the blastocyst stage was not different between treatments (14.1% v. 14.3%). Further work is needed to improve embryonic development, but reduced polyspermic penetration is an important step in the optimization of the PPM system for in vitro porcine embryo production.

Intracellular adenosine triphosphate and glutathione concentrations in oocytes from first estrous, multi-estrous, and testosterone-treated gilts.

Cytoplasmic maturation refers to a variety of cellular changes that must occur in the oocyte in order to progress through subsequent fertilization and embryonic development. Intracellular concentrations of ATP (ATPi) or glutathione (GSHi), indicative of metabolic activity or the ability of the oocyte to form a male pronucleus and cope with cellular stress, respectively, have been used as markers of cytoplasmic maturation in vitro. In the current study, our objective was to determine if concentrations of ATPi and GSHi in oocytes recovered from three groups of gilts were associated with known differences in developmental competence within these populations. In vivo matured oocytes were surgically recovered 36-38 h after the onset of estrus from first estrous gilts, multi-estrous gilts, and multi-estrous gilts receiving testosterone (1mg/2 ml per day; day 13 to estrus, day 0=day of estrus). Concentrations of ATPi and GSHi were determined using a bioluminescent somatic cell assay kit (luciferin-luciferase reaction) and the dithiobisnitrobenzoic acid (DTNB)-glutathione reductase recycling reaction, respectively. There were no differences (P>0.05) between ATPi concentrations in oocytes from the three groups (1.52 +/- 0.10, 1.51 +/- 0.11, 1.56 +/- 0.11pmol per oocyte). In contrast, oocytes from multi-estrous gilts had higher (P<0.05) concentrations of GSHi (31.53 +/- 1.66 to 33.67 +/- 2.30 pmol per oocyte) than oocytes from first estrous gilts (25.07 +/- 0.82). Administration of testosterone did not affect (P>0.05) GSHi concentrations in oocytes from multi-estrous gilts. Differences in developmental potential between the three groups of gilts were apparently not due to different concentrations of ATPi. However, GSHi concentrations were higher in oocytes from multi-estrous gilts, suggesting that reduced developmental potential of oocytes from first-estrus gilts may be related to insufficient amounts of GSHi. The beneficial effect of exogenous testosterone on the percentage of embryos surviving early gestation does not appear to be due to increased GSHi. Of the numerous potential markers of developmental potential, two were examined in the current study, and GSHi appeared to be useful for assessing porcine oocytes.

Gonadotropin exposure, salt storage and storage duration affect penetration of domestic cat oocytes by homologous spermatozoa.

Salt-stored domestic cat oocytes are routinely used to study sperm function in domestic and nondomestic felids. Our objectives were to assess the effects of in vitro maturation (IVM), salt storage and storage duration on penetration of domestic cat oocytes by homologous spermatozoa. In Experiment 1, domestic cat spermatozoa were coincubated with fresh immature oocytes, salt-stored (2-3 weeks) immature oocytes, or salt-stored (2-3 weeks) IVM oocytes matured in Minimum Essential Medium containing 0.1IU FSH and 0.1IU LH/ml (IVM1) or 0.5IU FSH and 2.2IULH/ml (IVM2). In Experiment 2, all oocytes were matured (IVM2) and inseminated fresh or after salt storage for 2-3 weeks, 2-3 months or 9 months. In Experiment 1, penetration of the outer zona pellucida (OZP) was greater (P<0.05) in salt-stored IVM2 oocytes than in salt-stored immature oocytes, whereas penetration of salt-stored IVM1 oocytes was intermediate (P>0.05). In Experiment 2, penetration of the OZP and inner zona pellucida (IZP) was higher (P<0.05) in fresh IVM2 oocytes than in salt-stored oocytes, and a higher (P<0.05) proportion of oocytes had IZP sperm after 2-3 weeks of storage than after 2-3 months. Penetration of the perivitelline space was higher (P<0.05) in fresh IVM2 oocytes than in oocytes stored for 2-3 weeks or 2-3 months. These results suggest that oocyte penetration is improved by IVM, but is impaired by exposure to salt-storage solution and prolonged storage duration.

Exposure to androgens during in vitro maturation does not affect the developmental potential of porcine oocytes.

Administration of exogenous androgens to pigs during the period of follicular development has been shown to positively affect ovulation rate and embryonic survival. The mechanisms of these actions are not known, but may include direct effects of androgens on the cumulus-oocyte complex (COC). The objective of this experiment was to assess the effects on embryonic development in vitro of exposure of COC to 0.26 and 2.6 microM testosterone (T) or dihydrotestosterone (DHT) during IVM. For IVM, COC were cultured for 44-46 h in protein-free tissue culture medium (TCM) 199 containing 10 IU/ml hCG and eCG and 10 ng/ml EGF. Oocytes were then stripped of cumulus cells, coincubated with 1 x 10(5) sperm/ml in modified TALP for 6 h, and cultured for 8 days in NCSU23. The proportions of oocytes that cleaved (Day 2) or developed to the morula (Day 6) or blastocyst (Day 6-8) stage were not different (P > 0.20) between oocytes exposed to androgens and oocytes not exposed to androgens. These results indicate that exposure to androgens during IVM does not affect the ability of oocytes to cleave or develop up to the blastocyst stage in vitro.

Sperm cryopreservation in endangered felids: developing linkage of in situ-ex situ populations.

Many of the world's cat species face growing threats to their continued survival in nature. For some species, managed captive populations may provide a reservoir for future reintroduction or genetic augmentation. Because most zoo populations are derived from small founder sizes and are subject to loss of genetic variation over time, periodic infusion of founder alleles is necessary to avoid the dire consequences of inbreeding. Collection and freezing of semen from free-living nondomestic felids offers a viable option for introducing founder genes into captive populations without removal of animals from the wild. The effective application of this strategy requires established protocols for safely capturing and anaesthetising wild cats coupled with suitable methods of semen recovery, processing and cryopreservation under field conditions. In small-sized non-domestic felids, the general feasibility of this approach is being explored in two studies of black-footed cats and Pallas' cats. Two factors - relatively low sperm numbers per ejaculate and compromised status of frozen-thawed cat spermatozoa - suggest that in vitro fertilisation (IVF) and embryo transfer present the most efficient use of this limiting resource in small-sized cats. Our studies with captive felids have explored alternative methods of sperm cryopreservation that are adaptable to field situations and shown that frozen-thawed spermatozoa from Pallas' cats, ocelots, and fishing cats exhibit adequate function to fertilise heterologous and/or homologous oocytes in vitro. Most recently, we investigated the fertilising capacity of frozen-thawed spermatozoa obtained from wild Pallas' cats in Mongolia. Combined with improved methods for embryo culture and transfer in small cat species, sperm banking in situ will facilitate introduction of new founders into captive populations without causing further depletion of their wild counterparts. As one component of holistic conservation programs, including ongoing support of field ecology studies in range countries, this reproductive strategy serves to further strengthen linkages among imperiled ex situ and in situ cat populations.

Increased ovulation rate in gilts treated with dihydrotestosterone.

Treatment with testosterone increases ovulation rate in pigs. The present study was conducted to examine the effects of 5alpha-dihydrotestosterone (DHT), a non-aromatizable androgen receptor ligand, on ovulation rate and amounts of androgen receptor and FSH receptor mRNAs in postpubertal gilts. In Expt 1, ovulation rate in response to daily i.m. injections of 0, 6, 60 or 600 microg DHT kg(-1) body weight from day 13 of the oestrous cycle (day 0 = day 1 of oestrus) to the following oestrus increased with each dose of DHT (P < 0.05). The mean increase in number of corpora lutea ranged from approximately three to 17 over the three dosages of DHT. In Expt 2, gilts treated daily with 60 microg DHT kg(-1) body weight during the early follicular phase (from day 13 to day 16), coincident with follicular recruitment, or the late follicular phase (day 17 to oestrus), had higher (P < 0.05) rates of ovulation compared with gilts that received vehicle, and were not different from gilts treated with DHT from day 13 to oestrus. Percentage recovery of day 3 embryos was not altered when gilts were treated from day 13 to day 16 or from day 17 to oestrus; however, treatment of gilts with DHT from day 13 to oestrus decreased recovery of day 3 (Expt 1) or day 11 (Expt 2) conceptuses. Daily administration of 6 microg DHT kg(-1) body weight to gilts from day 13 of the oestrous cycle to the following oestrus (Expt 3) did not affect the relative amounts of androgen receptor mRNA, but increased (P < 0.05) the amounts of FSH receptor mRNA in preovulatory follicles as determined by RT-PCR. The results of these experiments indicate that androgens may regulate ovulation rate in gilts. One of the roles of androgens might be regulation of the amounts of FSH receptor mRNA in ovarian follicles.

Urinary progesterone in free-ranging red howler monkeys (Alouatta seniculus): preliminary observations of the estrous cycle and gestation.

The goals of this study were to develop and validate a radioimmunoassay (RIA) for measurement of unconjugated progesterone (P) concentrations in the urine of red howler monkeys (Alouatta seniculus) and to use urinary P profiles to characterize the reproductive cycle of this species. Analysis of P profiles from two females provided a preliminary estimate of the length of the estrous cycle (mean days +/- S.E.M. = 29.5 +/- 1.5; n = 2), and indicated that one female red howler copulated throughout two apparent estrous cycles. Urinary P concentrations during two confirmed pregnancies (211.8 +/- 29.7 ng P/ml) were higher (P < 0.05) than during the luteal phase (77.4 +/- 10.6 ng P/ml; n = 4) of the cycle.

Effect of macromolecule supplementation during in vitro maturation of goat oocytes on developmental potential.

In vitro maturation (IVM) of goat oocytes with serum-supplemented media results in oocytes with reduced developmental potential. The objective of this study was to develop a defined medium for IVM of goat oocytes that better supports subsequent embryonic development. Cumulus oocyte complexes (COC) were matured for 18-20 hr in: Experiment (1), tissue culture medium 199 (TCM199) with 10% (v/v) goat serum or modified synthetic oviduct fluid maturation medium (mSOFmat) with 2.5, 8.0, or 20.0 mg/ml bovine serum albumin (BSA); Experiment (2), mSOFmat with 4.0, 8.0, 12.0, or 16.0 mg/ml BSA; or Experiment (3), 1.0 mg/ml polyvinyl alcohol (PVA; control), 4.0 mg/ml BSA, 0.5 mg/ml hyaluronate plus 0.5 mM citrate, or hyaluronate, citrate, and BSA. Mature COC were coincubated for 20-22 hr with 12-15 x 10(6) sperm/ml in modified Brackett and Oliphant (mBO) medium. Embryos were cultured for a total of 7 days in G1/2, and evaluated for cleavage, and blastocyst development, hatching, and total cell numbers. In the first experiment, more (P < 0.05) blastocysts developed per cleaved embryo following maturation in mSOFmat with 2.5 or 8.0 mg/ml BSA than with 20.0 mg/ml BSA or TCM199 with 10% goat serum. The various concentrations of BSA used in the second experiment did not affect (P > 0.05) any of the developmental endpoints examined. In the third experiment, developmental potential of oocytes matured with PVA or hyaluronate with citrate was not different (P > 0.05) from oocytes matured in the presence of BSA. These results demonstrate that developmentally competent goat oocytes can be matured under defined conditions.